Aminopyridyl-substituted phenyl acetamides as protease inhibitors

ABSTRACT

Phenyl acetamide compounds are described, including compounds of 
                 
 
or a solvate, hydrate or pharmaceutically acceptable salt thereof; wherein R 3 -R 6 , R 11 , B, Y and W are set forth in the specification. The compounds of the invention are potent inhibitors of proteases, especially trypsin-like serine proteases, such as thrombin and factor Xa. Compositions for inhibiting loss of blood platelets, inhibiting formation of blood platelet aggregates, inhibiting formation of fibrin, inhibiting thrombus formation, and inhibiting embolus formation are described. Other uses of compounds of the invention are as anticoagulants either embedded in or physically linked to materials used in the manufacture of devices used in blood collection, blood circulation, and blood storage, such as catheters, blood dialysis machines, blood collection syringes and tubes, blood lines and stents. Additionally, the compounds can be detectably labeled and employed for in vivo imaging of thrombi.

The present application is a divisional of U.S. patent application Ser.No. 09/971,000, filed Oct. 5, 2001 now U.S. Pat. No. 6,521,663, whichclaims priority to Provisional Application No. 60/238,132, filed Oct. 6,2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel compounds that function asproteolytic enzyme inhibitors, and particularly to a new class ofthrombin inhibitors.

2. Related Art

Proteases are enzymes that cleave proteins at single, specific peptidebonds. Proteases can be classified into four generic classes: serine,thiol or cysteinyl, acid or aspartyl, and metalloproteases (Cuypers etal., J. Biol. Chem. 257:7086 (1982)). Proteases are essential to avariety of biological activities, such as digestion, formation anddissolution of blood clots, reproduction and the immune reaction toforeign cells and organisms. Aberrant proteolysis is associated with anumber of disease states in man and other mammals. The human neutrophilproteases, elastase and cathepsin G, have been implicated ascontributing to disease states marked by tissue destruction. Thesedisease states include emphysema, rheumatoid arthritis, corneal ulcersand glomerular nephritis. (Barret, in Enzyme Inhibitors as Drugs,Sandier, ed., University Park Press, Baltimore, (1980)). Additionalproteases such as plasmin, C-1 esterase, C-3 convertase, urokinase,plasminogen activator, acrosin, and kallikreins play key roles in normalbiological functions of mammals. In many instances, it is beneficial todisrupt the function of one or more proteolytic enzymes in the course oftherapeutically treating a mammal.

Serine proteases include such enzymes as elastase (human leukocyte),cathepsin G, plasmin, C-1 esterase, C-3 convertase, urokinase,plasminogen activator, acrosin, chymotrypsin, trypsin, thrombin, factorXa and kallikreins.

Human leukocyte elastase is released by polymorphonuclear leukocytes atsites of inflammation and thus is a contributing cause for a number ofdisease states. Cathepsin G is another human neutrophil serine protease.Compounds with the ability to inhibit the activity of these enzymes areexpected to have an anti-inflammatory effect useful in the treatment ofgout, rheumatoid arthritis and other inflammatory diseases, and in thetreatment of emphysema. Chymotrypsin and trypsin are digestive enzymes.Inhibitors of these enzymes are useful in treating pancreatitis.Inhibitors of urokinase and plasminogen activator are useful in treatingexcessive cell growth disease states, such as benign prostatichypertrophy, prostatic carcinoma and psoriasis.

The serine protease thrombin occupies a central role in hemostasis andthrombosis, and as a multifactorial protein, induces a number of effectson platelets, endothelial cells, smooth muscle cells, leukocytes, theheart, and neurons. Activation of the coagulation cascade through eitherthe intrinsic pathway (contact activation) or the extrinsic pathway(activation by exposure of plasma to a non-endothelial surface, damageto vessel walls or tissue factor release) leads to a series ofbiochemical events that converge on thrombin. Thrombin cleavesfibrinogen ultimately leading to a hemostatic plug (clot formation),potently activates platelets through a unique proteolytic cleavage ofthe cell surface thrombin receptor (Coughlin, Seminars in Hematology31(4):270-277 (1994)), and autoamplifies its own production through afeedback mechanism. Thus, inhibitors of thrombin function havetherapeutic potential in a host of cardiovascular and non-cardiovasculardiseases.

Factor Xa is another serine protease in the coagulation pathway. FactorXa associates with factor Va and calcium on a phospholipid membranethereby forming a prothrombinase complex. This prothrombinase complexthen converts prothrombin to thrombin (Claeson, Blood Coagulation andFibrinolysis 5:411-436 (1994); Harker, Blood Coagulation andFibrinolysis 5 (Suppl 1):S47-S58 (1994)). Inhibitors of factor Xa arethought to offer an advantage over agents that directly inhibit thrombinsince direct thrombin inhibitors still permit significant new thrombingeneration (Lefkovits and Topol, Circulation 90(3):1522-1536 (1994);Harker, Blood Coagulation and Fibrinolysis 5 (Suppl 1):S47-S58 (1994)).

In vivo diagnostic imaging methods for intravascular thrombi have beenpreviously reported. These imaging methods use compounds that aredetectably labeled with radioactive or paramagnetic atoms. For example,platelets labeled with the gamma emitter, In-111, can be employed as animaging agent for detecting thrombi (Thakur, M. L. et al., Thromb Res.9:345 (1976); Powers et al., Neurology 32:938 (1982)). The thrombolyticenzyme streptokinase labeled with Tc-99m has been proposed as an imagingagent (Wong, U.S. Pat. No. 4,418,052 (1983)). The fibrin-binding domainsof Staphylococcus aureus derived protein A labeled with the gammaemitters, I-125 and I-131, have been proposed as imaging agents (Pang,U.S. Pat. No. 5,011,686 (1991)). Monoclonal antibodies havingspecificity for fibrin (in contrast to fibrinogen) and labeled withTc-99m have been proposed as imaging agents (Berger et al., U.S. Pat.No. 5,024,829 (1991); Dean et al., U.S. Pat. No. 4,980,148 (1990)). Theuse of the paramagnetic contrasting agent, gadoliniumdiethylenetriaminepentaacetic acid in magnetic resonance imaging ofpatients treated by thrombolysis for acute myocardial infarction hasbeen reported (De Roos, A. et al., Int. J. Card. Imaging 7:133 (1991)).Radiolabeled and paramagnetically labeled alpha-ketoamide derivativeshave also been proposed as thrombus imaging agents (Abelman et al., U.S.Pat. No. 5,656,600).

A need continues to exist for non-peptidic compounds that are potent andselective protease inhibitors, and which possess greater bioavailabilityand fewer side-effects than currently available protease inhibitors.Accordingly, new classes of potent protease inhibitors, characterized bypotent inhibitory capacity and low mammalian toxicity, are potentiallyvaluable therapeutic agents for a variety of conditions, includingtreatment of a number of mammalian proteolytic disease states.

SUMMARY OF THE INVENTION

The present invention is directed to novel aminopyridinyl-,aminoguanidinyl-, and alkoxyguanidinyl-substituted phenyl acetamideshaving Formula I (below). Also provided are processes for preparingcompounds of Formula I. The novel compounds of the present invention arepotent inhibitors of proteases, especially trypsin-like serineproteases, such as chymotrypsin, trypsin, thrombin, plasmin and factorXa. Certain of the compounds exhibit antithrombotic activity via direct,selective inhibition of thrombin, or are intermediates useful forforming compounds having antithrombotic activity. Also provided aremethods of inhibiting or treating aberrant proteolysis in a mammal andmethods of treating thrombosis, ischemia, stroke, restenosis orinflammation in a mammal by administering an effective amount of acompound of Formula I.

The invention includes a composition for inhibiting loss of bloodplatelets, inhibiting formation of blood platelet aggregates, inhibitingformation of fibrin, inhibiting thrombus formation, and inhibitingembolus formation in a mammal, comprising a compound of the invention ina pharmaceutically acceptable carrier. These compositions may optionallyinclude anticoagulants, antiplatelet agents, and thrombolytic agents.The compositions can be added to blood, blood products, or mammalianorgans in order to effect the desired inhibitions.

Also provided are methods of inhibiting or treating aberrant proteolysisin a mammal, and methods for treating myocardial infarction; unstableangina; stroke; restenosis; deep vein thrombosis; disseminatedintravascular coagulation caused by trauma, sepsis or tumor metastasis;hemodialysis; cardiopulmonary bypass surgery; adult respiratory distresssyndrome; endotoxic shock; rheumatoid arthritis; ulcerative colitis;induration; metastasis; hypercoagulability during chemotherapy;Alzheimer's disease; Down's syndrome; fibrin formation in the eye; andwound healing. Other uses of compounds of the invention are asanticoagulants either embedded in or physically linked to materials usedin the manufacture of devices used in blood collection, bloodcirculation, and blood storage, such as catheters, blood dialysismachines, blood collection syringes and tubes, blood lines and stents.

The invention also includes a method for reducing the thrombogenicity ofa surface in a mammal by attaching to the surface, either covalently ornoncovalently, a compound of the invention.

In another aspect, the present invention includes compositions which areuseful for in vivo imaging of thrombi in a mammal, comprising a compoundof the present invention which is capable of being detected outside thebody. Preferred are compositions comprising a compound of the presentinvention and a detectable label, such as a radioactive or paramagneticatom.

In another aspect, the present invention provides diagnosticcompositions which are useful for in vivo imaging of thrombi in amammal, comprising a pharmaceutically acceptable carrier and adiagnostically effective amount of a compound or composition of thepresent invention.

In another aspect, the present invention includes methods which areuseful for in vivo imaging of thrombi in a mammal.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Compounds of the present invention include compounds of Formula I:

or a solvate, hydrate or pharmaceutically acceptable salt thereof;wherein:

-   W is hydrogen, R¹, R¹OC(O), R¹C(O), R¹(CH₂)_(s)NHC(O), R¹S(O)₂, or    (R¹)₂CH(CH₂)_(s)NHC(O), wherein s is 0-4;-   R¹ is    -   R²,    -   R²(CH₂)_(t)C(R¹²)₂, where t is 0-3, and each R¹² can be the same        or different,    -   (R²)₂(OR¹²)CH(CH₂)_(p), where p is 1-4,    -   (R²)₂(OR¹²)C(CH₂)_(p), where p is 1-4,    -   R²C(R¹²)₂(CH₂)_(t), wherein t is 0-3, and each R¹² can be the        same or different, wherein (R¹²)₂ can also form a ring with C        represented by C₃₋₉ cycloalkyl,    -   R²CF₂C(R¹²)₂(CH₂)_(q), wherein q is 0-2, and each R¹² can be the        same or different, wherein (R¹²)₂ can also form a ring with C        represented by C₃₋₉cycloalkyl,    -   R²CH₂C(R¹²)₂(CH₂)_(q), wherein q is 0-2, and each R¹² can be the        same or different, wherein (R¹²)₂ can also form a ring with C        represented by C₃₋₉ cycloalkyl,    -   (R²)₂CH(CH₂)_(r), where r is 0-4 and each R² can be the same or        different, and wherein (R²)₂ can also form a ring with CH        represented by C₃₋₉ cycloalkyl, C₇₋₁₂ bicylic alkyl, C₁₀₋₁₆        tricylic alkyl, or a 5- to 7-membered mono- or bicyclic        heterocyclic ring which can be saturated or unsaturated, and        which contains from one to three heteroatoms selected from the        group consisting of N, O and S,    -   R²O(CH₂)_(p), wherein p is 2-4,    -   (R²)₂CF(CH₂)_(r), wherein r is 0-4 and each R² can be the same        different, wherein (R²)₂ can also form a ring with C represented        by C₃₋₉ cycloalkyl, C₇₋₁₂ bicyclic alkyl, C₁₀₋₁₆ tricyclic        alkyl, or a 5- to 7-membered mono- or bicyclic heterocyclic ring        which can be saturated or unsaturated, and which contains from        one to three heteroatoms selected from the group consisting of        N, O and S,    -    where s is 0 or 1, or    -   R²CF₂C(R¹²)₂;-   R² is    -   phenyl, naphthyl, or biphenyl, each of which is unsubstituted or        substituted with one or more of C₁₋₄ alkyl, C₁₋₄ alkoxy,        halogen, hydroxy, CF₃, OCF₃, COOH, CONH₂, or SO₂NH₂,    -   a 5- to 7-membered mono- or a 9- to 10-membered bicyclic        heterocyclic ring or non-heterocyclic ring which can be        saturated or unsaturated, wherein the heterocyclic ring contains        from one to four heteroatoms selected from the group consisting        of N, O and S, and wherein the heterocyclic or non-heterocyclic        ring is unsubstituted or substituted with halogen or hydroxy,    -   C₁₋₁₂ alkyl, unsubstituted or substituted with one or more of        hydroxy, COOH, amino, optionally C₁₋₃ alkyl substituted aryl,        C₃₋₉ cycloalkyl, CF₃, N(CH₃)₂, heteroaryl, or heterocycloalkyl,    -   CF₃,    -   C₃₋₉ cycloalkyl, unsubstituted or substituted with aryl,    -   C₇₋₁₂ bicyclic alkyl, or    -   C₁₀₋₁₆ tricyclic alkyl;-   Y is —NH— or —O—;-   R³, R⁴, R⁵ and R⁶ are independently hydrogen, alkyl, cycloalkyl,    alkenyl, alkynyl, optionally substituted aryl, optionally    substituted aralkyl, optionally substituted heteroaryl, haloalkyl,    hydroxy, alkoxy, aryloxy, heteoraryloxy, halogen, haloalkoxy,    hydroxyalkyl, cyano, nitro, —CO₂R^(x), —CH₂OR^(x) or —OR^(x),    -   where R^(x), in each instance, is independently one of hydrogen,        C₁₋₁₂ alkyl or C₃₋₉ cycloalkyl wherein said C₁₋₁₂ alkyl or C₃₋₉        cycloalkyl groups may optionally have one or more unsaturations;-   R¹¹ is hydrogen, alkyl, or alkenyl;-   R¹² is    -   hydrogen or halogen,    -   phenyl, naphthyl, or biphenyl, each of which is unsubstituted or        substituted with one or more of C₁₋₄ alkyl, C₁₋₄ alkoxy,        halogen, hydroxy, CF₃, OCF₃, COOH, or CONH₂,    -   a 5- to 7-membered mono- or a 9- to 10-membered bicyclic        heterocyclic ring which can be saturated or unsaturated, and        which contains from one to four heteroatoms selected from the        group consisting of N, O and S,    -   C₁₋₁₂ alkyl, unsubstituted or substituted with one or more of        hydroxy, COOH, amino, C₆₋₁₄ aryl, heteroaryl, or        heterocycloalkyl,    -   CF₃,    -   C₃₋₉ cycloalkyl,    -   C₇₋₁₂ bicyclic alkyl, or    -   C₁₀₋₁₆ tricyclic alkyl;-   B is selected from the group consisting of:    wherein    -   R⁷, R⁸, R⁹, and R¹⁰ are independently hydrogen, alkyl, aralkyl,        aryl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl,        dialkylaminoalkyl or carboxyalkyl;        -   or R⁷ and R⁸ are taken together to form —(CH₂)_(u)—, where u            is 2 to 7, preferably 2 to 5, while R⁹ and R¹⁰ are defined            as above;        -   or R⁹ and R¹⁰ are taken together to form —(CH₂)_(v)—, where            v is 2 to 7, preferably 2 to 5, while R⁷ and R⁸ are defined            as above;        -   or R⁷ and R⁹ are taken together to form —(CH₂)_(y)—, where y            is 0 (a bond) or 1 to 7, preferably 0-4, while R⁸ and R¹⁰            are defined as above;    -   X is —O—, —NR¹⁸—, or —CH═N— (where N is bonded to NR¹³) where        R¹⁸ is hydrogen, alkyl, cycloalkyl or aryl, wherein said alkyl,        cycloalkyl or aryl are optionally substituted with amino,        monoalkylamino, dialkylamino, alkoxy, hydroxy, carboxy,        alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, aryl,        heteroaryl, acylamino, cyano or trifluoromethyl;    -   R^(a), R^(b) and R^(c) are independently hydrogen, alkyl,        hydroxy, alkoxy, aryloxy, aralkoxy, alkoxycarbonyloxy, cyano or        —CO₂R^(w), where R^(w) is C₁₋₁₂ alkyl, C₃₋₉ cycloalkyl, C₆₋₁₄        aryl, C₆₋₁₄ar(C₁₋₁₂) alkyl,    -    where R^(e) and R^(f) are independently hydrogen, C₁₋₆ alkyl,        C₂₋₆ alkenyl or C₆₋₁₄ aryl, R⁸ is hydrogen, C₁₋₆ alkyl, C₂₋₆        alkenyl or C₆₋₁₄ aryl, Rh is hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl        or C₆₋₁₄ aryl, and R¹ is C₆₋₁₄ar(C₁₋₁₂)alkyl or C₁₋₁₂ alkyl;    -   n is from zero to 8; and    -   m is from zero to 6;    -   R¹³ is hydrogen, alkyl, alkenyl, aralkyl, aryl, hydroxyalkyl,        aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl or        carboxyalkyl;    -   R¹⁴ and R¹⁵ are independently hydrogen, alkyl, cycloalkyl,        halogen or alkoxy; and    -   R¹⁶ and R¹⁷ are independently hydrogen, alkyl, hydroxy, alkoxy,        aryloxy, alkoxycarbonyl, cyano or —CO₂R¹, where R¹ is C₁₋₁₂        alkyl, C₃₋₉ cycloalkyl, C₆₋₁₄ aryl, C₆₋₁₄ar(C₁₋₁₂)alkyl,        halo(C₁₋₁₂)alkyl or    -    where R^(e), R^(f) and R^(g) are independently hydrogen or        C₁₋₁₂ alkyl.

Compounds within the scope of the present invention include those forwhich:

-   -   R³, R⁴, R⁵ and R⁶ are independently hydrogen, C₁₋₁₂ alkyl, C₃₋₉        cycloalkyl, halogen, C₂₋₂₀ alkenyl, C₂₋₂₀ alkynyl, optionally        substituted C₆₋₁₄ aryl, optionally substituted        C₆₋₁₄ar(C₁₋₁₂)alkyl, optionally substituted heteroaryl,        halo(C₁₋₁₂)alkyl, C₁₋₁₂ alkoxy, C₆₋₁₄ aryloxy, heteroaryloxy,        halo(C₁₋₂₀)alkoxy or hydroxy(C₁₋₁₂)alkyl;    -   R¹¹ is hydrogen, C₁₋₁₂ alkyl or C₂₋₂₀ alkenyl;    -   R⁷, R⁸, R⁹ and R¹⁰ are independently hydrogen, C₁₋₁₂ alkyl,        C₆₋₁₄ar(C₁₋₁₂)alkyl, C₆₋₁₄ aryl, hydroxy(C₁₋₁₂)alkyl,        amino(C₁₋₁₂)alkyl, mono(C₁₋₁₂)alkylamino(C₁₋₂)alkyl,        di(C₁₋₁₂)alkylamino(C₁₋₁₂)alkyl, or carboxy(C₁₋₁₂)alkyl;    -   R¹⁸ is C₁₋₂ alkyl, C₃₋₉ cycloalkyl or C₆₋₁₄ aryl, each of which        is optionally substituted with amino, mono(C₁₋₁₂)alkylamino,        di(C₁₋₁₂)alkylamino, C₁₋₂₀ alkoxy, hydroxy, carboxy, C₁₋₂₀        alkoxycarbonyl, C₆₋₁₄ aryloxy carbonyl, C₆₋₁₄        ar(C₁₋₂₀)alkoxycarbonyl, C₆₋₁₄ aryl, C₅₋₁₀ heteroaryl,        acylamino, cyano or trifluoromethyl;    -   R^(a), R^(b) and R^(c) are independently C₁₋₁₂ alkyl, C₁₋₂₀        alkoxy, C₆₋₁₄ aryloxy, C₆₋₁₄ar(C₁₋₂₀)alkoxy, or C₁₋₂₀        alkoxycarbonyloxy;    -   R¹³ is C₁₋₁₂ alkyl, C₁₋₂₀ alkoxy, C₆₋₁₄ aryloxy or C₁₋₂₀        alkoxycarbonyl;    -   R¹⁴ and R¹⁵ are independently C₁₋₁₂ alkyl, C₃₋₉ cycloalkyl or        C₁₋₂₀ alkoxy; and    -   R¹⁶ and R¹⁷ are independently C₁₋₁₂ alkyl, C₁₋₂₀ alkoxy, C₆₋₁₄        aryloxy or C₁₋₂₀ alkoxycarbonyl.

Preferred compounds of Formula I above are those for which Y is —NH— or—SO₂NH—.

A preferred subgenus of compounds of Formula I above are those for whichB is

where R⁷-R¹⁰, R¹³ and R^(a)-R^(c) are as defined above

Another preferred subgenus of compounds of Formula I above are those forwhich B is

where R⁹, R¹⁰ and R¹⁴-R¹⁷ are as defined above.

Preferred compounds of Formula I above are those for which W is R¹,where R¹ is R² and R² is either optionally substituted phenyl,optionally substituted naphthyl or C₁₋₇ alkyl substituted with aryl.

Preferred compounds of Formula I above are those for which R¹ isR²CF₂C(R¹²)₂(CH₂)_(q).

Preferred compounds of Formula I above are those for which R⁶ is C₁₋₆alkyl or halogen. More preferred compounds within the third preferredsubgenus are those for which R⁶ is methyl or chloro, including compoundsfor which R⁶ is chloro while R³ is fluoro.

Preferred compounds of Formula I above are those for which R¹¹ ishydrogen.

Preferred values of R^(a), R^(b) and R^(c) in Formula I areindependently hydrogen, hydroxy, C₁₋₆alkyl, C₁₋₆ alkoxy, cyano or—CO₂R^(w), where R^(w), in each instance, is preferably one ofC₁₋₄alkyl, C₁₋₇cycloalkyl or benzyloxycarbonyl. Suitable values ofR^(a), R^(b) and R^(c) include hydrogen, methyl, ethyl, propyl, n-butyl,hydroxy, methoxy, ethoxy, cyano, —CO₂CH₃, —CO₂CH₂CH₃ and —CO₂CH₂CH₂CH₃.In the most preferred embodiments, R^(a), R^(b) and R^(c) are eachhydrogen.

Also preferred at R^(a), R^(b) and R^(c) is the group —CO₂R^(w), whereR^(w) is one of

where R^(e)-R^(f) are defined as above. When R^(a), R^(b) and R^(c) are—CO₂R^(c), where R^(w) is one of one of these moieties, the resultingcompounds are prodrugs that possess desirable formulation andbioavailability characteristics. A preferred value for each of R^(e),R^(f) and R^(h) is hydrogen, R^(g) is methyl, and preferred values forR^(i) include benzyl and tert-butyl.

Preferred compounds are those of Formula I, where R⁷, R⁸, R⁹ and R¹⁰ areindependently one of hydrogen, C₁₋₆ alkyl, C₆₋₁₀ ar(C₁₋₆)alkyl, C₆₋₁₀aryl, C₂₋₁₀ hydroxyalkyl or C₂₋₇ carboxyalkyl. Useful values of R⁷, R⁸,R⁹ and R¹⁰ include hydrogen, methyl, ethyl, propyl, n-butyl, benzyl,phenylethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl,2-carboxymethyl, 3-carboxyethyl and 4-carboxypropyl. Additionalpreferred compounds are those where R⁷ and R⁸ or R⁹ and R¹⁰ are takentogether to form —(CH₂)_(y)— where y is 2.

Preferred compounds when X is NR¹⁸ are those wherein R¹⁸ is hydrogen orC₁₋₆ alkyl, optionally substituted by one, two or three, preferably one,of amino, monoalkylamino, dialkylamino, alkoxy, hydroxy, alkoxycarbonyl,aryloxycarbonyl, aralkoxycarbonyl, carboalkoxy, phenyl, cyano,trifluoromethyl, acetylamino, pyridyl, thiophenyl, furyl, pyrrolyl orimidazolyl.

Suitable values of R¹⁸ include hydrogen, methyl, ethyl, propyl, n-butyl,benzyl, phenethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl,carboxymethyl and carboxyethyl.

Most preferred compounds are those where X is oxygen.

R⁶ can represent hydrogen, C₁₋₃ alkyl, halogen, or C₁₋₂ alkoxy. R⁶ ispreferably C₁₋₃ alkyl, e.g., methyl, or halogen, e.g., chlorine, bromineor fluorine.

R³, R⁴, R⁵ and R⁶ can independently represent hydrogen, hydroxy, C₁₋₃alkyl, halogen, or C₁₋₂ alkoxy. Preferably R³ is fluorine and hydroxy.

Preferred values of n in Formula I include from zero to 6, morepreferably from zero to 4, and most preferably zero, 1 or 2. Preferredvalues of m include from zero to 4, more preferably zero, 1, 2 or 3.

It is also to be understood that the present invention is considered toinclude stereoisomers as well as optical isomers, e.g. mixtures ofenantiomers as well as individual enantiomers and diastereomers, whicharise as a consequence of structural asymmetry in selected compounds ofthe present series. The compounds of the present invention may also havepolymorphic crystalline forms, with all polymorphic crystalline formsbeing included in the present invention.

The compounds of Formula I may also be solvated, especially hydrated.Hydration may occur during manufacturing of the compounds orcompositions comprising the compounds, or the hydration may occur overtime due to the hygroscopic nature of the compounds.

Certain compounds within the scope of Formula I are derivatives referredto as prodrugs. The expression “prodrug” denotes a derivative of a knowndirect acting drug, which derivative has enhanced deliverycharacteristics and therapeutic value as compared to the drug, and istransformed into the active drug by an enzymatic or chemical process.Useful prodrugs are those where R^(a), Rb, R^(c) and/or R^(d) are—CO₂R^(w), where R^(w) is defined above. See, U.S. Pat. No. 5,466,811and Saulnier et al., Bioorg. Med. Chem. Lett. 4:1985-1990 (1994).

When any variable occurs more than one time in any constituent or inFormula I, its definition on each occurrence is independent of itsdefinition at every other occurrence. Also, combinations of substituentsand/or variables are permissible only if such combinations result instable compounds.

In another aspect, the present invention includes compositions which areuseful for in vivo imaging of thrombi in a mammal, comprising a compoundof the present invention which is capable of being detected outside thebody. Preferred are compositions comprising a compound of the presentinvention and a detectable label, such as a radioactive or paramagneticatom.

In another aspect, the present invention provides diagnosticcompositions which are used for in vivo imaging of thrombi in a mammal,comprising a pharmaceutically acceptable carrier and a diagnosticallyeffective amount of a compound or composition of the present invention.

In another aspect, the present invention includes methods which areuseful for in vivo imaging of thrombi in a mammal.

According to a preferred aspect, useful compounds are those wherein theR¹ substituent is substituted with a detectable label, such as aradioactive iodine atom, such as I-125, I-131 or I-123. In this aspect,R¹ is preferably phenyl, having a para I-123, para I-125 or para I-131substitution, or benzyl, having a meta I-123, meta I-125 or meta I-131substitution.

The detectable label can also be a radioactive or paramagnetic chelatein which a suitable ligand (L) is attached to an R¹ substituent, eitherdirectly or via a divalent linking group A″. Alternatively, the group—A″—L substitutes for the group W in Formula I. By suitable ligand ismeant an organic moiety that is capable of chelating a radioactive orparamagnetic metal ion.

In these compounds, the divalent linking group A″ includes groups thatare capable of covalently bonding with a free amino group and thechelating means. For example, A″ may be —C(═S)—, —C(═O)—, (═NH)—(CH₂)₆(═NH)—, C(═O)+CH₂)₆—(═O),

and the like.

Also, in the compounds represented by Formula I, the chelating ligand,L, includes groups capable of covalently bonding to or noncovalentlybinding to either a radioactive or paramagnetic atom. The chelatingmeans including those which are customarily used for complexingradioactive or paramagnetic atoms. These include chelating meanscontaining 3 to 12, preferably 3 to 8, methylene phosphonic acid groups,methylene carbohydroxamic acid groups, carboxyethylidene groups, orespecially carboxymethylene groups, which are bonded to a nitrogen atom.If only one or two of the acid groups are bonded to a nitrogen atom,then that nitrogen is bonded to another nitrogen atom having such groupsby an optionally substituted ethylene group or by up to four separatedethylene units separated by a nitrogen or oxygen or sulfur atom.Preferred as a completing means isdiethylenetrimine-N,N,N′,N″,N″-pentaacetic acid (DTPA). DTPA is wellknown in the art as a chelating means for the radioactive atomsindium-111 (In-111), technetium-99m (Tc-99m), and the paramagnetic atomgadolinium (Gd). Khaw, et al., Science 209:295 (1980); Paik C. H. etal., U.S. Pat. No. 4,652,440 (1987); Gries, H. et al., U.S. Pat. No.4,957,939 (1990). A preferred chelating ligand, L, is1-(p-aminobenzyl)-diethylenetriaminepentaacetic acid. Also included aschelating means are compounds which contain sulfhdryl or amine moieties,the total of which in any combination is at least four. These sulfhydrylor amine moieties are separated from each other by at least two atomswhich can be either carbon, nitrogen, oxygen, or sulfur. Especiallypreferred for chelating means, L, is metallothionein which is well knownin the art as a chelating means for Tc-99m.

The term “alkyl” as employed herein by itself or as part of anothergroup refers to both straight and branched chain radicals of up to 12carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl,isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl,2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl. Preferably, alkylis 1 to 6 carbon atoms.

The term “alkenyl” is used herein to mean a straight or branched chainradical of 2-20 carbon atoms, unless the chain length is limitedthereto, including, but not limited to, ethenyl, 1-propenyl, 2-propenyl,2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Preferably, thealkenyl chain is 2 to 10 carbon atoms in length, more preferably, 2 to 8carbon atoms in length most preferably from 2 to 4 carbon atoms inlength.

The term “alkynyl” is used herein to mean a straight or branched chainradical of 2-20 carbon atoms, unless the chain length is limitedthereto, wherein there is at least one triple bond between two of thecarbon atoms in the chain, including, but not limited to, acetylene,1-propylene, 2-propylene, and the like. Preferably, the alkynyl chain is2 to 10 carbon atoms in length, more preferably, 2 to 8 carbon atoms inlength, most preferably from 2 to 4 carbon atoms in length.

In all instances herein where there is an alkenyl or alkynyl moiety as asubstituent group, the unsaturated linkage, i.e., the vinylene oracetylene linkage, is preferably not directly attached to a nitrogen,oxygen or sulfur moiety.

The term “alkoxy” is used herein to mean a straight or branched chainradical of 1 to 20 carbon atoms, unless the chain length is limitedthereto, bonded to an oxygen atom, including, but not limited to,methoxy, ethoxy, n-propoxy, isopropoxy, and the like. Preferably thealkoxy chain is 1 to 10 carbon atoms in length, more preferably 1 to 8carbon atoms in length.

The term “aryl” as employed herein by itself or as part of another grouprefers to monocyclic or bicyclic aromatic groups containing from 6 to 14carbons in the ring portion, preferably 6-10 carbons in the ringportion, such as phenyl, naphthyl or tetrahydronaphthyl.

The term “heteroaryl” as employed herein refers to groups having 5 to 14ring atoms; 6, 10 or 14 π electrons shared in a cyclic array; andcontaining carbon atoms and 1, 2 or 3 oxygen, nitrogen or sulfurheteroatoms (where examples of heteroaryl groups are: thienyl,benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl,isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl, phenoxathiinyl,2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl,indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl,phthalazinyl, naphthyridinyl, quinazolinyl, cinnolinyl, pteridinyl,4αH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl,perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,isoxazolyl, furazanyl and phenoxazinyl groups).

The term “aralkyl” or “arylalkyl” as employed herein by itself or aspart of another group, refers to C₁₋₁₂ alkyl, preferably C₁₋₆ alkyl,groups as discussed above having an aryl substituent, such as benzyl,phenylethyl or 2-naphthylmethyl.

The term “cycloalkyl” as employed herein by itself or as part of anothergroup, refers to cycloalkyl groups containing 3 to 9 carbon atoms,preferably 3 to 7 carbon atoms. Typical examples are cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl andcyclononyl.

The term “C₇₋₁₂ bicyclic alkyl” is intended to includebicyclo[2.2.1]heptyl (norbornyl), bicyclo[2.2.2]octyl,1,1,3-trimethylbicyclo[2.2.1]heptyl (bornyl), and the like.

The term “C₁₀₋₁₆ tricyclic alkyl” is intended to include tricyclo[5, 2,1, 0^(2,6)] decyl, adamantyl, and the like.

The term “halogen” or “halo” as employed herein by itself or as part ofanother group refers to chlorine, bromine, fluorine or iodine withchlorine and fluorine being preferred.

The term “monoalkylamine” as employed herein by itself or as part ofanother group refers to an amino group which is substituted with onealkyl group having from 1 to 12, preferably 1 to 6, carbon atoms.

The term “dialkylamine” as employed herein by itself or as part ofanother group refers to an amino group which is substituted with twoalkyl groups, each having from 1 to 12, preferably 1 to 6, carbon atoms.

The term “hydroxyalkyl” as employed herein refers to any of the abovealkyl groups substituted by one or more hydroxyl moieties.

The term “carboxyalkyl” as employed herein refers to any of the abovealkyl groups substituted by one or more carboxylic acid moieties.

The term “heterocycle” or “heterocyclic ring”, as used herein exceptwhere noted, represents a stable 5- to 7-membered mono- or bicyclic orstable 7- to 10-membered bicyclic heterocyclic ring system, any ring ofwhich may be saturated or unsaturated, and which consists of carbonatoms and from one to three heteroatoms selected from the groupconsisting of N, O and S, and wherein the nitrogen and sulfurheteroatoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized, and including any bicyclic group in which anyof the above-defined heterocyclic rings is fused to a benzene ring.Especially useful are rings containing one oxygen or sulfur, one tothree nitrogen atoms, or one oxygen or sulfur combined with one or twonitrogen atoms. The heterocyclic ring may be attached at any heteroatomor carbon atom which results in the creation of a stable structure.Examples of such heterocyclic groups include piperidinyl, piperazinyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl,azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl,isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,quinuclidinyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl,benzimidazolyl, thiadiazoyl, benzopyranyl, benzothiazolyl, benzoxazolyl,furyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzothienyl,thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, andoxadiazolyl. Morpholino is the same as morpholinyl.

The term “heteroatom” is used herein to mean an oxygen atom (“O”), asulfur atom (“S”) or a nitrogen atom (“N”). It will be recognized thatwhen the heteroatom is nitrogen, it may form an NR^(a)R^(b) moiety,wherein R^(a) and R^(b) are, independently from one another, hydrogen orC₁ to C₈ alkyl, or together with the nitrogen to which they are bound,form a saturated or unsaturated 5-, 6-, or 7-membered ring.

Schemes 1-8 outline a synthetic route to compounds of Formula I

In Scheme 1, an acetic acid side chain is introduced onto a benzene ringby reaction of a fluorinated nitrobenzene 1, such as1,2,3-trifluoro-4-nitrobenzene, with the metal salt of a substituted ornon-substituted malonate ester, such as diethyl malonate, in a suitablesolvent such as tetrahydrofuran (THF), followed by acid hydrolysis andsubsequent decarboxylation upon heating, to produce compound 2(Yokomoto, M, W., et al., EP published application No. 0 470 578 A1(1991)). The carboxyl group of 2 is converted to a hydroxyl group undertypical reducing conditions, such as borane (BH₃)-THF complex and sodiumborohydride (NaBH₄), in a suitable solvent such as THF, to give alcohol3 (Yokomoto, M, W., et al., ibid). Introduction of a suitablefunctionality para to the nitro group in the ring is achieved byaromatic nucleophilic substitution of the fluoride in compound 3 with asuitable nucleophile, such as tert-butylamine, in suitable solvents suchas dimethyl sulfoxide (DMSO) and toluene under reflux, to affordcompound 4 (Yokomoto, M, W., et al., ibid). The nitrogen protectinggroup, such as tert-butyl, in compound 4 is removed under standardconditions, such as concentrated hydrochloric acid (HCl) under reflux,to give compound 5 (Yokomoto, M, W., et al., ibid). The hydroxyl groupof compound 5 is masked with a suitable protecting group, such asacetyl, under standard conditions well known in the art (Greene, T. W.,and Wuts, P. G. M., Protecting Groups in Organic Synthesis, 2^(nd) ed.,John Wiley and Sons, Inc., New York (1991)), such as acetyl chloride indichloromethane (DCM) in the presence of base such as triethylamine ordiisopropylethylamine (DIEA), to give compound 6. Coupling of anactivated carbonyl compound ACOCl with compound 6 in a suitable solvent,such as DCM, produces compound 7.

In Scheme 2, reduction of arylnitro compound 7 under typical conditions,such as catalytic hydrogenation with hydrogen in the presence ofpalladium on activated carbon in ethanol or methanol, gives arylamine 8.The acetyl protecting group of compound 8 is removed (in order toincrease the solubility of the compound prior to the amino groupmanipulation) by hydrolysis under basic conditions, such as aqueouspotassium carbonate (K₂CO₃) solution in methanol, to free the protectedhydroxyl group, giving compound 9. The desired R⁶ is introduced into thecenter scaffold of compound 9 by a Sandmeyer-type reaction ((a)Gunstone, F. D., et al., Org. Syn. Collect Vol. 1, Wiley, New York, N.Y.(1941), p. 170; (b) Yokomoto, M, W., et al., EP published applicationNo. 0 470 578 A1 (1991)) with suitable reagents, such as sodium nitrite(NaNO₂) and HCl followed by copper (I) chloride (CuCl), or bysubstitutive deamination (Doyle, M. P., et al. J. Org. Chem. 42:2426(1977)) with suitable reagents, such as tert-butylnitrite (t-BuONO) andcopper (II) chloride (CuCl₂), to give compound 10. The amino group ofarylamine 9 can be converted to a methyl group under carbon—carboncoupling conditions in the presence of a palladium catalyst through anarenediazonium salt intermediate (Kikukawa, K., et al., J. Org. Chem.48:1333 (1983)). Compound 10 in turn, is reduced with a suitablereducing agent, such as BH₃, to generate desired fragment WY of compound11 where Y is —NH—. Oxidation of 11 with an oxidizing agent, such assulfur trioxide pyridine complex (SO₃ pyridine) in DCM, yields aldehyde12. Construction of the center and left fragment of the target compoundis finally achieved by further oxidation of the aldehyde 12 tocarboxylic acid 13 under suitable oxidation conditions, such as sodiumchlorite (NaClO₂) in the presence of sodium dihydrogenphosphate(NaH₂PO₄) and DMSO (Dalcanale, E., et al., J. Org. Chem. 51:567 (1986)).

In Scheme 3, acid 13 is coupled with a suitable amine 14, such asprotected O-guanidinyl amine (Tianbao Lu, et al., WO 99/26926 (1999)),or aminopyridinyl amine (Sanderson, P. E., et al., WO 97/01338 (1997))in the presence of a typical peptide coupling reagent, such as Castro'sreagent (BOP), and a base, such as DIEA, in a suitable solvent, such asN,N-dimethylformamide (DMF), to produce amide 15. Optionally, theprotecting groups, such as tert-(butoxy)carbonyl (Boc), can be removedunder typical deprotection conditions, such as trifluoroacetic acid(TFA) solution in DCM when B is O-guanidine, or HCl solution in1,4-dioxane when B is aminopyridine, to generate free O-guanidine, oraminopyridine, respectively.

In Scheme 4, the phenylacetic acid derivative 16 is nitrated in the metaposition of the benzene ring using standard conditions, such as 96%nitric acid in conc. sulfuric acid (Sindelar et al., Coll. Czechoslov.Chem. Commun. 42:2231 (1977)), to give the nitro compound 17. Thecarboxylic acid group of compound 17 is then protected using standardconditions well known in the art (Greene, T. W. and Wuts, P. G. M.,Protective Groups in Organic Synthesis, 2^(nd) ed., John Wiley and Sons,New York (1991)), such as conversion to the ester by reaction withoxalyl chloride followed by alcohol POH, to afford ester 18 (where P isa typical carboxylic acid protecting group). Reduction of the nitrogroup is accomplished using a suitable reagent, such as tin (II)chloride, in an appropriate solvent, such as ethanol, and the resultingamine 19 is reacted with an acylating agent (W=R¹C(O)) or asulfonylating agent (W=R¹S(O)₂), such as benzylsulfonyl chloride, and asuitable base, such as N-methylmorpholine, in a solvent, such as DCM, toprovide the N-substituted-aminophenylacetate 20 (Y=—NH—). The carboxylicacid group is deprotected using standard conditions well known in theart (Greene, T. W. and Wuts, P. G. M., Protective Groups in OrganicSynthesis, 2^(nd) edition, John Wiley and Sons, New York (1991)), suchas hydrolysis with aqueous hydroxide, to give acid 13 (Y═—NH—). This isthen coupled with amine 14 and deprotected, as in Scheme 3, to producephenylacetamide 15 (Y═NH).

In Scheme 5, nitrophenylacetic acid 17 is coupled to an aminoalcohol 21,such as ethanolamine, using a standard peptide coupling procedure, suchas in Scheme 3, to give alcohol 22. The alcohol is converted to theprotected alkoxyamine by coupling to N-hydroxyphthalimide using standardreagents (Mitsunobu, O., Synthesis 1:1 (1981)), such astriphenylphosphine and diethylazodicarboxylate, in a suitable solvent,such as THF, to afford compound 23, which is then converted to aniline24 under typical reducing conditions, such as hydrogenation overpalladium(0) on carbon, in a suitable solvent, such as ethanol. Theamine is then acylated or sulfonylated as in Scheme 4 to giveintermediate 25, and the alkoxyamine deprotected using standardconditions well known in the art (Greene, T. W. and Wuts, P. G. M.,Protective Groups in Organic Synthesis, 2^(nd) edition, John Wiley andSons, New York (1991)), such as aqueous methylamine in ethanol/THF.Guanidinylation of the resulting alkoxyamine 26 is accomplished with astandard guanidinylation reagent, such asN,N′-bis(tert-butoxycarbonyl)-S-methylthiourea (Bergeron, R. J. andMcManis, J. S., J. Org. Chem. 52:1700 (1987)) or N-R^(a)-N′-R^(b),R^(c)-1H-pyrazole-1-carboxamidine (Bernatowicz, M. S. et al. TetrahedronLett. 34:3389 (1993)), and the guanidine optionally deprotected as inScheme 3, to provide final target 27.

In scheme 6, the ketone, aldehyde (R¹¹=H), or carboxylic acid (R¹¹=OH)starting material 28 is reduced with a suitable reagent, such asborane-THF, to give alcohol 29, which is then converted to a betterleaving group by reaction with a sulfonyl chloride, such asmethanesulfonyl chloride, in a suitable solvent, such as DCM, to producecompound 30. The sulfonate is displaced by cyanide under standardconditions, such as potassium cyanide in refluxing acetonitrile, to givenitrile 31, which is then hydrolyzed with a typical reagent, such asaqueous hydroxide. Coupling of the resulting acid 17 with amine 14 isaccomplished as in scheme 3 to give intermediate 32, and the nitro groupis reduced as in Scheme 4 or 5 to afford aniline 33. This is acylated orsulfonylated as in Scheme 4 and the guanidine optionally deprotected asin Scheme 3 to give the final target 15 (Y=NH).

In Scheme 7, nitrophenol 34 is alkylated with allylic halide 35 and asuitable base, such a cesium carbonate, in a polar aprotic solvent, suchas DMF, giving intermediate 36, which is then converted to compound 37via the aromatic Claisen rearrangement by heating. The phenol isprotected using typical reagents, such as benzyl bromide and cesiumcarbonate, in a solvent, such as DMF, to give 38 (where P is a typicalhydroxyl protecting group) and the nitro group is reduced as in Scheme 4or 5 to produce aniline 39. Aniline 39 is converted to intermediate 40as in Scheme 4 and the alkene is oxidatively cleaved using standardconditions, such as sodium periodate and osmium tetraoxide indioxane/water followed by Jones reagent, to provide acid 41. This isthen coupled to amine 14, the guanidine optionally deprotected as inScheme 3, and the phenol group optionally deprotected using standardconditions, such as hydrogenation over palladium (0) on carbon, in asuitable solvent, such as ethanol, to produce the target compound 42.

In Scheme 8, the mono-protected catechol 43 is sulfonylated with areagent W—Cl, such as meta-toluenesulfonyl chloride, in a solvent, suchas DCM, in the presence of a base, such as triethylamine, givingcompound 44. The protecting group is removed using standard conditions,such as boron tribromide in DCM, and the resulting phenol 45 isalkylated with allylic halide 35 to give 46, rearranged to phenol 47,and protected to afford intermediate 48 as in Scheme 7. The alkene isoxidatively cleaved using standard conditions, such as sodium periodateand ruthenium(III) chloride in acetonitrile/water (Ashby, E. C. andGoel, A. B., J. Org. Chem. 46:3936 (1981)) followed by Jones reagent,giving acid 49, which is then coupled with amine 14 and optionallydeprotected as in Schemes 3 and 7 to afford target compound 50.

The pharmaceutically-acceptable salts of the compounds of Formula I (inthe form of water- or oil-soluble or dispersible products) include theconventional non-toxic salts or the quaternary ammonium salts which areformed, e.g., from inorganic or organic acids or bases. Examples of suchacid addition salts include acetate, adipate, alginate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, sulfate, tartrate, thiocyanate, tosylate,trifluoroacetate, and undecanoate. Base salts include ammonium salts,alkali metal salts such as sodium and potassium salts, alkaline earthmetal salts such as calcium and magnesium salts, salts with organicbases such as dicyclohexylamine salts, N-methyl-D-glucamine, and saltswith amino acids such as arginine, lysine, and so forth, including saltswith a guanidinyl moiety. Also, the basic nitrogen-containing groups maybe quaternized with such agents as lower alkyl halides, such as methyl,ethyl, propyl, and butyl chlorides, bromides and iodides; dialkylsulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates; longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides; aralkyl halides like benzyl and phenethyl bromidesand others. Preferred acids for forming acid addition salts include HCl,acetic acid and trifluoroacetic acid.

The compounds of the present invention represent a novel class of potentinhibitors of metallo, acid, thiol and serine proteases. Examples of theserine proteases inhibited by compounds within the scope of theinvention include leukocyte neutrophil elastase, a proteolytic enzymeimplicated in the pathogenesis of emphysema; chymotrypsin and trypsin,digestive enzymes; pancreatic elastase, and cathepsin G, achymotrypsin-like protease also associated with leukocytes; thrombin andfactor Xa, proteolytic enzymes in the blood coagulation pathway.Inhibition of thermolysin, a metalloprotease, and pepsin, an acidprotease, are also contemplated uses of compounds of the presentinvention. The compounds of the present invention are preferablyemployed to inhibit trypsin-like proteases.

For their end-use application, the potency and other biochemicalparameters of the enzyme-inhibiting characteristics of the compounds ofthe present invention are readily ascertained by standard biochemicaltechniques known to those of skill in the art. For example, an end useapplication of the compounds that inhibit chymotrypsin and trypsin is inthe treatment of pancreatitis. Actual dose ranges for their specificend-use application will, of course, depend upon the nature and severityof the disease state of the patient or animal to be treated, asdetermined by the attending diagnostician. It is expected that a usefuldose range will be about 0.01 to 10 mg per kg per day for an effectivetherapeutic effect.

Compounds of the present invention that are distinguished by theirability to inhibit thrombin may be employed for a number of therapeuticpurposes. As thrombin inhibitors, compounds of the present inventioninhibit thrombin production. Therefore, these compounds are useful forthe treatment or prophylaxis of states characterized by abnormal venousor arterial thrombosis involving either thrombin production or action.These states include, but are not limited to, deep vein thrombosis;disseminated intravascular coagulopathy which occurs during septicshock, viral infections and cancer; myocardial infarction; stroke;coronary artery bypass; fibrin formation in the eye; hip replacement;and thrombus formation resulting from either thrombolytic therapy orpercutaneous transluminal coronary angioplasty (PCTA). Other usesinclude the use of said thrombin inhibitors as anticoagulants eitherembedded in or physically linked to materials used in the manufacture ofdevices used in blood collection, blood circulation, and blood storage,such as catheters, blood dialysis machines, blood collection syringesand tubes, and blood lines. The compounds of the present invention mayalso be used as an anticoagulant in extracorporeal blood circuits.

Stents have been shown to reduce restenosis, but are thrombogenic. Astrategy for reducing the thrombogenicity of stents is to coat, embed,adsord or covalently attach a thrombin-inhibiting agent to the stentsurface. The compounds of the present invention can be employed for thispurpose. Compounds of the invention can be attached to, or embeddedwithin soluble and/or biodegradeable polymers as and thereafter coatedonto stent materials. Such polymers can include polyvinylpyrrolidone,polyhydroxy-propylmethacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues, polylactic acid, polyglycolic acid,copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross linked or amphipathicblock copolymers of hydrogels. See European Application 761 251,European Application 604,022, Canadian Patent No. 2,164,684 and PCTPublished Applications Nos. WO 96/11668, WO 96/32143 and WO 96/38136.

By virtue of the effects of thrombin on a host of cell types, such assmooth muscle cells, endothelial cells and neutrophils, the compounds ofthe present invention find additional use in the treatment orprophylaxis of adult respiratory distress syndrome; inflammatoryresponses; wound healing; reperfusion damage; atherosclerosis; andrestenosis following an injury such as balloon angioplasty, atherectomy,and arterial stent placement.

The compounds of the present invention may be useful in treatingneoplasia and metastasis as well as neurodegenerative diseases, such asAlzheimer's disease and Parkinson's disease.

When employed as thrombin inhibitors, the compounds of the presentinvention may be administered in an effective amount within the dosagerange of about 0.1 to about 500 mg/kg, preferably between 0.1 to 10mg/kg body weight, on a regimen in single or 2-4 divided daily doses.

When employed as inhibitors of thrombin, the compounds of the presentinvention may be used in combination with thrombolytic agents such astissue plasminogen activator, streptokinase, and urokinase.Additionally, the compounds of the present invention may be used incombination with other antithrombotic or anticoagulant drugs such as,but not limited to, fibrinogen antagonists and thromboxane receptorantagonists.

The thrombin inhibitors may also be coupled with soluble polymers astargetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxy-propylmethacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the thrombininhibitors may be coupled to a class of biodegradable polymers useful inachieving controlled release of a drug, for example, polylactic acid,polyglycolic acid, copolymers of polylactic and polyglycolic acid,polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydihydropyrans, polycyanoacrylates and cross linked oramphipathic block copolymers of hydrogels.

Human leucocyte elastase is released by polymorphonuclear leukocytes atsites of inflammation and thus is a contributing cause for a number ofdisease states. Compounds of the present invention are expected to havean anti-inflammatory effect useful in the treatment of gout, rheumatoidarthritis and other inflammatory diseases, and in the treatment ofemphysema. The leucocyte elastase inhibitory properties of compounds ofthe present invention are determined by the method described below.Cathepsin G has also been implicated in the disease states of arthritis,gout and emphysema, and in addition, glomerulonephritis and lunginfestations caused by infections in the lung. In their end-useapplication the enzyme inhibitory properties of the compounds of FormulaI are readily ascertained by standard biochemical techniques that arewell-known in the art.

The Cathepsin G inhibitory properties of compounds within the scope ofthe present invention are determined by the following method. Apreparation of partially purified human Cathepsin G is obtained by theprocedure of Baugh et al., Biochemistry 15: 836 (1979). Leukocytegranules are a major source for the preparation of leukocyte elastaseand cathepsin G (chymotrypsin-like activity). Leukocytes are lysed andgranules are isolated. The leukocyte granules are extracted with 0.20 Msodium acetate, pH 4.0, and extracts are dialyzed against 0.05 M Trisbuffer, pH 8.0 containing 0.05 M NaCl overnight at 4° C. A proteinfraction precipitates during dialysis and is isolated by centrifugation.This fraction contains most of the chymotrypsin-like activity ofleukocyte granules. Specific substrates are prepared for each enzyme,namely N-Suc-Ala-Ala-Pro-Val-p-nitroanilide andSuc-Ala-Ala-Pro-Phe-p-nitroanilide. The latter is not hydrolyzed byleukocyte elastase. Enzyme preparations are assayed in 2.00 mL of 0.10 MHepes buffer, pH 7.5, containing 0.50 M NaCl, 10% dimethylsulfoxide and0.0020 M Suc-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. Hydrolysisof the p-nitroanilide substrate is monitored at 405 nm and at 25° C.

Useful dose range for the application of compounds of the presentinvention as neutrophil elastase inhibitors and as Cathepsin Ginhibitors depend upon the nature and severity of the disease state, asdetermined by the attending diagnostician, with a range of 0.01 to 10mg/kg body weight, per day, being useful for the aforementioned diseasestates.

Compounds of the present invention that inhibit urokinase or plasminogenactivator are potentially useful in treating excessive cell growthdisease state. As such compounds of the present invention may also beuseful in the treatment of benign prostatic hypertrophy and prostaticcarcinoma, the treatment of psoriasis, and as abortifacients. For theirend-use application, the potency and other biochemical parameters of theenzyme inhibiting characteristics of compounds of the present inventionare readily ascertained by standard biochemical techniques well known inthe art. Actual dose ranges for this application will depend upon thenature and severity of the disease state of the patient or animal to betreated as determined by the attending diagnostician. It is to beexpected that a general dose range will be about 0.01 to 10 mg per kgper day for an effective therapeutic effect.

Additional uses for compounds of the present invention include analysisof commercial reagent enzymes for active site concentration. Forexample, chymotrypsin is supplied as a standard reagent for use inclinical quantitation of chymotrypsin activity in pancreatic juices andfeces. Such assays are diagnostic for gastrointestinal and pancreaticdisorders. Pancreatic elastase is also supplied commercially as areagent for quantitation of α₁-antitrypsin in plasma. Plasmaα₁-antitrypsin increases in concentration during the course of severalinflammatory diseases, and α₁-antitrypsin deficiencies are associatedwith increased incidence of lung disease. Compounds of the presentinvention can be used to enhance the accuracy and reproducibility ofthese assays by titrametric standardization of the commercial elastasesupplied as a reagent. See, U.S. Pat. No. 4,499,082.

Protease activity in certain protein extracts during purification ofparticular proteins is a recurring problem which can complicate andcompromise the results of protein isolation procedures. Certainproteases present in such extracts can be inhibited during purificationsteps by compounds of the present invention, which bind tightly tovarious proteolytic enzymes.

The pharmaceutical compositions of the invention can be administered toany animal that can experience the beneficial effects of the compoundsof the invention. Foremost among such animals are humans, although theinvention is not intended to be so limited.

The pharmaceutical compositions of the present invention can beadministered by any means that achieve their intended purpose. Forexample, administration can be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, or ocular routes.Alternatively, or concurrently, administration can be by the oral route.The dosage administered will be dependent upon the age, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired.

In addition to the pharmacologically active compounds, the newpharmaceutical preparations can contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries thatfacilitate processing of the active compounds into preparations that canbe used pharmaceutically.

The pharmaceutical preparations of the present invention aremanufactured in a manner that is, itself, known, for example, by meansof conventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

For compositions of the present invention suitable for administration toa human, the term “excipient” is meant to include, but not be limitedby, those excipients described in the Handbook of PharmaceuticalExcipients, American Pharmaceutical Association, 2^(nd) Ed. (1994),which is herein incorporated by reference in its entirety. Suitableexcipients are, in particular, fillers such as saccharides, for example,lactose or sucrose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, for example, tricalcium phosphate or calciumhydrogen phosphate, as well as binders, such as, starch paste, using,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxy-propylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents can be added, such as, the above-mentionedstarches and also carboxymethyl-starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as, sodiumalginate. Auxiliaries are, above all, flow-regulating agents andlubricants, for example, silica, talc, stearic acid or salts thereof,such as, magnesium stearate or calcium stearate, and/or polyethyleneglycol. Dragee cores are provided with suitable coatings that, ifdesired, are resistant to gastric juices. For this purpose, concentratedsaccharide solutions can be used, which may optionally contain gumarabic, talc, polyvinyl pyrrolidone, polyethylene glycol, and/ortitanium dioxide, lacquer solutions and suitable organic solvents orsolvent mixtures. In order to produce coatings resistant to gastricjuices, solutions of suitable cellulose preparations, such as,acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate,are used. Dye stuffs or pigments can be added to the tablets or drageecoatings, for example, for identification or in order to characterizecombinations of active compound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as, glycerol or sorbitol. Thepush-fit capsules can contain the active compounds in the form ofgranules that may be mixed with fillers such as lactose, binders such asstarches, and/or lubricants such as talc or magnesium stearate and,optionally, stabilizers. In soft capsules, the active compounds arepreferably dissolved or suspended in suitable liquids, such as, fattyoils or liquid paraffin. In addition, stabilizers may be added.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts, alkaline solutions and cyclodextrin inclusioncomplexes. Especially preferred alkaline salts are ammonium saltsprepared, for example, with Tris, choline hydroxide, Bis-Tris propane,N-methylglucamine, or arginine. One or more modified or unmodifiedcyclodextrins can be employed to stabilize and increase the watersolubility of compounds of the present invention. Useful cyclodextrinsfor this purpose are disclosed in U.S. Pat. Nos. 4,727,064, 4,764,604,and 5,024,998.

In addition, suspensions of the active compounds as appropriate oilyinjection suspensions can be administered. Suitable lipophilic solventsor vehicles include fatty oils, for example, sesame oil, or syntheticfatty acid esters, for example, ethyl oleate or triglycerides orpolyethylene glycol-400 (the compounds are soluble in PEG-400). Aqueousinjection suspensions can contain substances that increase the viscosityof the suspension, for example, sodium carboxymethyl cellulose,sorbitol, and/or dextran. Optionally, the suspension may also containstabilizers.

Compounds of Formula I can be labeled with radioactive iodine by usingan exchange reaction. Exchange of hot iodine for cold iodine is wellknown in the art. Alternatively, a radio iodine labeled compound can beprepared from the corresponding bromo compound via a tributylstannylintermediate. See, U.S. Pat. No. 5,122,361, herein incorporated byreference.

The present invention also includes compositions which are useful for invivo imaging of thrombi in a mammal, wherein the compositions arecomprised of a compound of Formula I complexed with a radioactive atom.

For the compounds of Formula I, suitable radioactive atoms includeCo-57, Cu-67, Ga-67, Ga-68, Ru-97, Tc-99m, In-111, In-113m, Hg-197,Au-198, and Pb-203. Some radioactive atoms have superior properties foruse in radiochemical imaging techniques. In particular, technetium-99m(Tc-99m) is an ideal radioactive atom for imaging because of its nuclearproperties. Rhenium-186 and -188 also have gamma emission which allowsit to be imaged. Preferred compositions contain the radioactive atom,Tc-99m.

The compounds of Formula I can be labeled by any of the many techniquesknown in the art to provide a composition of the present invention. Forexample, these compounds can be labeled through a chelating agent suchas diethylene-triaminepentaacetic acid (DTPA) or metallothionein, bothof which can be covalently attached to the compound of Formula I.

In general, the compositions of the present invention containingtechnetium-99m are prepared by forming an aqueous mixture oftechnetium-99m and a reducing agent and a water-soluble ligand, and thencontacting the mixture with a compound of the present inventionrepresented by Formula I. For example, the imaging compounds of thisinvention are made by reacting technetium-99m (in an oxidized state)with the compounds of the present invention having a chelating means inthe presence of a reducing agent to form a stable complex betweentechnetium-99m in a reduced state (IV or V valence state).

One embodiment of the composition of the present invention is preparedby labeling a compound of Formula I having a DTPA chelating means withtechnetium-99m. This may be accomplished by combining a predeterminedamount (as 5 μg to 0.5 mg) of compound of the present invention with anaqueous solution containing citrate buffer and stannous reducing agent,then adding freshly eluted sodium pertechnetate containing apredetermined level of radioactivity (as 15 mCi). After allowing anincubation of the mixture at room temperature, the reaction mixture isloaded into a shielded syringe through a sterile filter (0.2-0.22micron), then is dispensed into 0.9% saline for injection, if desired.

Another embodiment of the compositions of the present invention isprepared by labeling a compound of Formula I having a metallothioneinchelating means with technetium-99m. This may be accomplished bycombining aqueous sodium pertechnetate-99m with aqueous stannousglucoheptonate to form a soluble complex of technetium-99m (in reducedstate) with two glucoheptonate molecules, then combining this solutionwith a compound of the Formula I having a metallothionein attachedthereto. After incubating the mixture for a period of time and underconditions which allow for an exchange of the technetium-99m from theglucoheptonate complex to the metallothionein of the compound of FormulaI, the technetium-labeled composition of the present invention isformed.

Reducing agents for use in the method are physiologically acceptable forreducing technetium-99m from its oxidized state to the IV or V valencestate or for reducing rhenium from its oxidized state. Reducing agentswhich can be used are stannous chloride, stannous fluoride, stannousglucoheptonate, stannous tartarate, and sodium dithionite. The preferredagents are stannous reducing agents, especially stannous chloride orstannous glucoheptonate. The amount of reducing agent is that amountnecessary to reduce the technetium-99m to provide for the binding to thechelating means of a compound of Formula I in this radioisotope'sreduced state. For example, stannous chloride (SnCl₂) is the reducingagent and can be used in range from 1-1,000 μg/mL.

Citric acid complexes with technetium-99m quickly to form a stabletechnetium-99m-citrate complex. Upon contact with a compound of FormulaI, substantially quantitative transfer of technetium-99m from itscitrate complex to the chelating means of the compound of Formula I isachieved rapidly and under mild conditions. The amount of citric acid(as sodium citrate) can range from about 0.5 mg/ml up to the amountmaximally soluble in the medium. Preferred amounts of citric acid rangefrom 15 to 30 μg/ml.

The amount of compound of Formula I having a chelating means can rangefrom 0.001 to about 3 mg/mL, preferably about 0.017 to about 0.15 mg/mL.Finally, technetium-99m in the form of pertechnetate can be used inamounts of preferably about 1-50 mCi. The amount of mCi per mg ofcompound of the present invention is preferably about 30-150.

The reaction between the compound of Formula I and the metalion-transfer ligand complex is preferably carried out in a aqueoussolution at a pH at which the compound of Formula I is stable. By“stable”, it is meant that the compound remains soluble and retains itsinhibitory activity against α-thrombin. Normally, the pH for thereaction will be from about 5 to 9, the preferred pH being above 6-8.The technetium-99m-citrate complex and a compound of Formula I areincubated, preferably at a temperature from about 20° C. to about 60°C., most preferably from about 20° C. to about 37° C., for a sufficientamount of time to allow transfer of the metal ion from the citratecomplex to the chelating means of the compound of Formula I. Generally,less than one hour is sufficient to complete the transfer reaction underthese conditions.

Alternative compositions of the present invention include an In-111labeled compound of the present invention.

The present invention also includes compositions of the compounds of thepresent invention which are useful for in vivo imaging of thrombi in amammal, comprised of a compound represented by Formula I complexed to aparamagnetic atom.

Preferred paramagnetic atoms are divalent or trivalent ions of elementswith an atomic number of 21 to 29, 42, 44 and 58 to 70. Suitable ionsinclude chromium(III), manganese(II), iron(III), iron(II), cobalt(II),nickel(II), copper(II), praseodymium(III), neodymium(III), samarium(III)and ytterbium(III). Because of their very strong magnetic moments,gadolinium(III), terbium(III), dysoprosium(III), holmium(III), anderbium(III) are preferred. Especially preferred for the paramagneticatom is gadolinium(III).

The compositions of the present invention may be prepared by combining acompound of Formula I with a paramagnetic atom. For example, the metaloxide or a metal salt (for example, nitrate, chloride or sulfate) of asuitable paramagnetic atom is dissolved or suspended in a mediumcomprised of water and an alcohol, such as methyl, ethyl or isopropylalcohol. This mixture is added to a solution of an equimolar amount ofthe compound of Formula I in a similar aqueous medium and stirred. Thereaction mixture may be heated moderately until the reaction iscompleted. Insoluble compositions formed may be isolated by filtering,while soluble compositions may be isolated by evaporation of thesolvent. If acid groups on the chelating means are still present in thecomposition of the present invention, inorganic or organic bases, andeven amino acids, may be added to convert the acidic complex into aneutral complex to facilitate isolation or purification of homogenouscomposition. Organic bases or basic amino acids may be used asneutralizing agents, as well as inorganic bases such as hydroxides,carbonates or bicarbonates of sodium, potassium or lithium.

The present invention also include diagnostic compositions which areuseful for in vivo imaging of thrombi in a mammal, comprising apharmaceutically acceptable carrier and a diagnostically effectiveamount of compositions derived from the compounds of Formula I.

The “diagnostically effective amount” of the composition required as adose will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific mammalunder consideration. These factors and their relationship to determiningthis dose are well known to skilled practitioners in the medialdiagnostic arts. Also, the diagnostically effective amount and method ofadministration can be tailored to achieve optimal efficacy but willdepend on such factors as weight, diet, concurrent medication and otherfactors which those skilled in the medical arts will recognize. In anyregard, the dose for imaging should be sufficient for detecting thepresence of the imaging agent at the site of a thrombus in question.Typically, radiologic imaging will require that the dose provided by thepharmaceutical composition position of the present invention be about 5to 20 μCi, preferably about 10 μCi. Magnetic resonance imaging willrequire that the dose provided be about 0.001 to 5 mmole/kg, preferablyabout 0.005 to 0.5 mmole/kg of a compound of Formula I complexed withparamagnetic atom. In either case, it is known in the art that theactual dose will depend on the location of the thrombus.

“Pharmaceutically acceptable carriers” for in vivo use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The pharmaceutical compositions of the present invention may beformulated with a pharmaceutically acceptable carrier to provide sterilesolutions or suspensions for injectable administration. In particular,injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspensions in liquid prior to injection, or as emulsions, Suitableexcipients are, for example, water, saline, dextrose, mannitol, lactose,lecithin, albumin, sodium glutamate, cysteine hydrochloride, or thelike. In addition, if desired, the injectable pharmaceuticalcompositions may contain minor amounts of nontoxic auxiliary substances,such as wetting agents, pH buffering agents, and the like. If desired,absorption enhancing preparations (e.g., liposomes) may be utilized.

The present invention also encompasses diagnostic compositions preparedfor storage or administration. These would additionally containpreservatives, stabilizers and dyes. For example, sodium benzoate,sorbic acid and esters of p-hydroxybenzoic acid may be added aspreservatives. Id. at 1449. In addition, antioxidants and suspendingagents may be used.

The in vivo imaging methods of the present invention also offer severaladvantages over previous imaging techniques for the detection ormonitoring of the presence, size, regression or increase of a thrombus.In particular, the present invention provides compounds, compositionsand diagnostic compositions that bind tightly to the thrombin associatedwith a thrombus and thereby reduce “background” due to circulatingradioactivity or paramagnetism arising from unbound imaging agent.Furthermore, in vivo imaging by intracoronary injection of thecompounds, compositions or diagnostic compositions of the presentinvention, is expected to be almost instantaneous since these imagingagents would saturate the thrombin bound to the thrombus immediately.

Accordingly, the present invention also includes methods for in vivoimaging of a thrombus in a mammal, comprising the steps of: (1)administering to a mammal a diagnostically acceptable amount of acompound, composition, or diagnostic composition of the presentinvention and (2) detecting a thrombus in a blood vessel.

The term “in vivo imaging” as used herein relates to methods of thedetection of a thrombus in a mammal, as well as the monitoring of thesize, location and number of thrombi in a mammal, as well as dissolutionor growth of the thrombus.

In employing the compounds, compositions or diagnostic compositions invivo by this method, “administering” is accomplished parenterally, ineither a systemic or local targeted manner. Systemic administration isaccomplished by injecting the compounds, compositions by diagnosticcompositions of the present invention into a convenient and accessiblevein or artery. This includes but is not limited to administration bythe ankecubutal vein. Local targeted administration is accomplished byinjecting the compounds, compositions or diagnostic compositions of thepresent invention proximal in flow to a vein or artery suspected tocontain thrombi distal to the injection site. This includes but is notlimited to direct injection into the coronary arterial vasculature toimage coronary thrombi, into the carotid artery to image thrombi in thecerebral vasculature, or into a pedal vein to image deep vein thrombosisof the leg.

Also, the manner of delivery of a composition of the present inventionto the site of a thrombus is considered within the scope of the term“administering”. For example, a compound represented by Formula I havinga chelating means attached thereto may be injected into the mammal,followed at a later time by the radioactive atom thereby forming in vivoat the site of the thrombus the composition comprising the compound offormula complexed to radioactive atom. Alternatively, a compositioncomprising the compound of formula complexed to radioactive atom may beinjected into the mammal.

The “diagnostically effective amount” of the compounds, compositions ordiagnostic compositions used in the methods of the present inventionwill, as previously mentioned, depend on the route of administration,the type of mammal being treated, and the physical characteristics ofthe specific mammal under treatment. These factors and theirrelationship to determining this dose are well known to skilledpractitioners in the medical diagnostic arts. In any regard, the dosefor in vivo imaging should be sufficient for detecting the presence ofthe imaging agent at the site of a thrombus in question. Typically,radiologic imaging will require that the dose provided by the diagnosticcomposition of the present invention be about 5 to 20 μCi, preferablyabout 10 μCi. Magnetic resonance imaging will require that the doseprovided by the diagnostic composition be about 0.001 to 5 mmole/kg,preferably about 0.005 to 0.5 mmole/kg of a compound of Formula Icomplexed with paramagnetic atom. In either case, it is known in the artthat the actual dose will depend on the location of the thrombus.

The detecting of a thrombus by imaging is made possible by the presenceof radioactive or paramagnetic atoms localized at such thrombus.

The radioactive atoms associated with the compositions and diagnosticcompositions of the present invention are preferably imaged using aradiation detection means capable of detecting gamma radiation, such asa gamma camera or the like. Typically, radiation imaging cameras employa conversion medium (wherein the high energy gamma ray is absorbed,displacing an electron which emits a photon upon its return to theorbital state), photoelectric detectors arranged in a spatial detectionchamber (to determine the position of the emitted photons), andcircuitry to analyze the photons detected in the chamber and produce animage.

The paramagnetic atoms associated with the compositions and diagnosticcompositions of the present invention are detected in magnetic resonanceimaging (MRI) systems. In such systems, a strong magnetic field is usedto align the nuclear spin vectors of the atoms in a patient's body. Thefield is disturbed by the presence of paramagnetic atoms localized at athrombus and an image of the patient is read as the nuclei return totheir equilibrium alignments.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered and obvious to those skilled in the art are within thespirit and scope of the invention.

EXAMPLES Example 1N-[2-(Amidinoaminooxy)ethyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetamideTrifluoroacetate Salt

1. Ethyl 2,2-difluoro-2-phenylacetate (Middleton, W., et al. J. Org.Chem. 42:2883 (1980)).

PhCF₂CO₂Et

A mixture of ethyl benzoylformate (12.5 g, 70.0 mmol) and(diethylamino)sulfur trifluoride (DAST, 18.5 mL, 140 mmol) was stirredfor 48 hours at ambient temperature, and then poured over ice. The oilformed was taken up into dichloromethane (DCM), washed with H₂O, driedover Na₂SO₄, concentrated, and filtered through a short column of silicagel eluting with 50% DCM/hexane. The filtrate was concentrated to givethe title compound (12.3 g, 88% yield) as a brown liquid. ¹H NMR (400MHz, CDCl₃) δ 7.65-7.63 (m, 2H), 7.52-7.43 (m, 3H), 4.30 (q, J=7.1 Hz,2H), 1.30 (t, J=7.1 Hz, 3H).

2. 2,2-Difluoro-2-phenylacetic Acid

PhCF₂CO₂H

A suspension of ethyl 2,2-difluoro-2-phenylacetate (6.0 g, 30 mmol), asprepared in the preceding step, in 1 N NaOH (36 mL, 36 mmol) was stirredat ambient temperature. After 36 hours, the reaction became almosthomogeneous. The mixture was acidified with 1N HCl (36 mL), andextracted with DCM twice. The extracts were combined, washed with H₂O,dried over Na₂SO₄, and concentrated to give the title compound (3.85 g,81% yield) as a pale yellow solid, that was used without furtherpurification.

3. 2,2-Difluoro-2-phenylacetyl Chloride

PhCF₂COCl

To a flask charged with 2,2-difluoro-2-phenylacetic acid (0.8 g, 5.06mmol), as prepared in the preceding step, under argon in an ice-bath wasadded oxalyl chloride (5 mL), and the reaction mixture stirred for 15min. A solution of dimethylformamide (DMF) (37 mg, 0.506 mmol) in DCM(0.5 mL) was added. After 2 hours, the ice-bath was removed, and themixture continued to stir for 1 hour. The solvents were evaporated, DCMwas added, and then evaporated in vacuo giving the title compound (0.88g, 98% yield), that was used immediately without further purification.

4. 2-(2,3-Difluoro-6-nitrophenyl)acetic Acid (Yokomoto, M, W., et al.1991, EP 0 470 578 A1)

To a suspension of NaH (11.3 g, 60% oil dispersion, 282 mmol) intetrahydrofuran (THF) (35 mL) in an ice-bath was added a solution ofdiethyl malonate (45.2 g, 42.9 mL, 282 mmol) in THF (70 mL) over aperiod of an hour so that the reaction temperature was kept below 20° C.Some white solid precipitated during the addition. To the above reactionmixture was added a solution of 1,2,3-trifluoro-4-nitrobenzene (25.0 g,141 mmol) in THF (35 mL) over a period of 1 hour so that the reactiontemperature was kept below 10° C. The ice-bath was removed and themixture was stirred at ambient temperature for 2 hours. Acetic acid (18mL) was added to the reaction solution, and THF was evaporated underreduced pressure. Chloroform (200 mL), H₂O (250 mL), and concentratedHCl (18 mL) were added. The organic layer was separated, concentrated,mixed with 4N HCl (45 mL) and acetic acid (35 mL), and refluxed for 14hours. The reaction mixture was allowed to cool to room temperature. Thesolid precipitated upon cooling was filtered off, washed withdiisopropyl ether, and dissolved in MeOH (70 mL). After treating withactive carbon, the solvent was evaporated, and the crystalline residuewashed with isopropyl ether, and filtered off to give the title compound(17.6 g, 58% yield) as a white solid. ¹H-NMR (400 MHz, CDCl₃) δ8.05-8.01 (m, 1H), 7.47 (dd, J=17.4, 8.9 Hz, 1H), 4.10 (s, 2H).

5. 2-(2,3-Difluoro-6-nitrophenyl)ethanol (Yokomoto, M, W., et al., EP 0470 578 A1 (1991))

To a mixture of NaBH₄ (3.60 g, 95.4 mmol) in THF (12 mL) cooled below10° C. was added a solution of 2-(2,3-difluoro-6-nitrophenyl)acetic acid(10.9 g, 50.2 mmol), as prepared in the preceding step, in THF (4 mL)over a period of 1 hour. To this mixture was added a solution of borontrifluoride diethyl etherate complex (16.5 mL, 131 mmol) in THF (24 mL)over a period of 1 hour, keeping the reaction temperature below 10° C.After the addition the reaction was continued to stir on ice for 15minutes, and then at ambient temperature for 20 minutes. To a mixture ofDCM (180 mL) and H₂O (140 mL) was added NaHCO₃ (15 g, 179 mmol). Thereaction mixture was slowly added to the above NaHCO₃ solution andstirred overnight. The organic layer was separated, dried over Na₂SO₄,and concentrated to give the title compound (10.1 g, 99% yield) as lightbrown oil. ¹H NMR (400 MHz, CDCl₃) δ 7.82 (dd, J=9.1, 4.4 Hz, 1H),7.27-7.18 (m, 1H), 3.95 (t, J=6.3 Hz, 2H), 3.30-3.27 (m, 2H), 1.82 (s,1H).

6. 2-{3-[(tert-Butyl)amino]-2-fluoro-6-nitrophenyl}ethanol (Yokomoto, M,W., et al. EP 0 470 578 A1 (1991)).

A mixture of 2-(2,3-difluoro-6-nitrophenyl)ethanol (6.00 g, 29.6 mmol),as prepared in the preceding step, tert-butylamine (18.6 mL, 1.77 mmol),DMSO (30 mL), and toluene (5 mL) was heated at reflux for 16 hours.After cooling to ambient temperature the brown solution was poured intoH₂O (300 mL), and the deposited yellow crystals were filtered and washedwith H₂O twice. The yellow solid was dissolved in CHCl₃ (70 mL), driedover Na₂SO₄, concentrated, and crystallized from hexane to give thetitle compound (4.70 g, 62% yield) as a yellow solid. ¹H NMR (400 MHz,CDCl₃) δ 7.92 (dd, J=9.3, 1.5 Hz, 1H), 6.79 (t, J=8.7 Hz, 1H), 4.69 (brs, 1H), 3.96 (dd, J=11.4, 5.9 Hz, 2H), 3.32 (dt, J=6.5, 3.1 Hz, 2H),1.75 (t, J=5.3 Hz, 1 H), 1.46 (s, 9H).

7. 2-(3-Amino-2-fluoro-6-nitrophenyl)ethanol (Yokomoto, M, W., et al. EP0 470 578 A1 (1991))

A solution of 2-{3-[(tert-butyl)amino]-2-fluoro-6-nitrophenyl}ethanol(3.9 g, 15 mmol), as prepared in the preceding step, in concentrated HCl(40 mL) was refluxed for 2 hours. After cooling to room temperature, themixture was extracted with ethyl acetate (6×50 mL). The extracts werecombined, washed with saturated NaHCO₃ (2 times) and brine, dried overNa₂SO₄, and concentrated to give the crude product as a solid. The solidwas triturated in hexane, filtered, and dried in high vacuum to producethe title compound (2.8 g, 93% yield) as a yellow solid. ¹H-NMR (400MHz, CD₃OD) δ 7.80 (dd, J=9.1, 1.5 Hz, 1H), 6.70 (t, J=9.0 Hz, 1H), 3.77(t, J=7.1 Hz, 2H), 3.26 (dt, J=7.3, 2.8 Hz, 2).

8. 2-(3-Amino-2-fluoro-6-nitrophenyl)ethyl Acetate

To a solution of DIEA (1.80 mL, 10.6 mmol) and2-(3-amino-2-fluoro-6-nitrophenyl)ethanol (0.88 g, 4.40 mmol), asprepared in the preceding step, in THF (10 mL) in an ice-bath was addeda solution of acetyl chloride (319 μL, 4.49 mmol) in THF (5 mL). Afterstirring for 1.5 hour, the ice-bath was removed and the mixture wascontinued to stir at ambient temperature overnight. Additional acetylchloride (63 μL, 0.88 mmol) was added, and the mixture was stirred foranother 16 hours. The solvents were removed, and the mixture waspartitioned between DCM and H₂O. The organic layer was separated, andthe aqueous layer was back-extracted with DCM. The organic layers werecombined, washed with H₂O (twice), dried over Na₂SO₄, concentrated, andflash chromatographed on silica gel eluting with EtOAc/DCM (0, 1, 2, and5%) to give the title compound (0.73 g, 69% yield) as a yellow solid.¹H-NMR (400 MHz, CDCl₃) δ 7.85 (dd, J=9.0, 1.5 Hz, 1H), 6.68 (t, J=8.9Hz, 1H), 4.38-4.35 (m, 4H), 3.38 (dt, J=6.6, 2.8 Hz, 2H), 2.03 (s, 3H).

9. 2-[3-(2,2-Difluoro-2-phenylacetylamino)-2-fluoro-6-nitrophenyl]ethylAcetate

To a solution of DIEA (1.49 mL, 8.55 mmol) and2-(3-amino-2-fluoro-6-nitrophenyl)ethyl acetate (690 mg, 2.85 mmol), asprepared in the preceding step, in DCM (6 mL) was added a solution of2,2-difluoro-2-phenylacetyl chloride (0.99 g, 5.20 mmol), as preparedaccording to the procedure of step 3 of Example 1, in DCM (3 mL). Afterstirring for 24 hours, the mixture was concentrated, and partitionedbetween DCM and H₂O. The organic layer was separated, and the aqueouslayer extracted with DCM. The organic layers were combined, washed withH₂O and brine, dried over Na₂SO₄, concentrated, and flashchromatographed on silica gel eluting with EtOAc/DCM (0, 2.5, and 5%) togive the title compound (1.04 g, 92% yield) as an orange oil. ¹H-NMR(400 MHz, CDCl₃) δ 8.49-8.43 (m, 2H), 7.87 (d, J=9.2 Hz, 1H), 6.68 (d,J=7.1 Hz, 2H), 7.55-7.49 (m, 3H), 4.35 (t, J=6.4 Hz, 2H), 3.37 (dt,J=6.3, 2.3 Hz, 2H), 2.01 (s, 3H).

10. 2-[2-Amino-5-(2,2-fluoro-2-phenylacetylamino)-6-fluorophenyl]ethylAcetate

A mixture of2-[3-(2,2-difluoro-2-phenylacetylamino)-2-fluoro-6-nitrophenyl]ethylacetate (0.84 g, 2.12 mmol), as prepared in the preceding step, andpalladium catalyst (226 mg, 10% on activated carbon, 0.212 mmol) inethanol (17 mL) was hydrogenated under a hydrogen balloon for 3.5 hours.The mixture was filtered through Celite (diatomaceous earth) and washedwith MeOH. The filtrate and washings were combined, concentrated, andflash chromatographed on silica gel eluting with EtOAc/DCM (5, 10, and20%) to give the title compound (0.713 g, 92% yield) as a white solid.¹H-NMR (400 MHz, CDCl₃) δ 8.10 (bs, 1H), 7.81 (t, J=8.7 Hz, 1H),7.68-7.66 (m, 2H), 7.52-7.44 (m, 3H), 6.45 (dd, J=8.8, 1.2 Hz, 1H), 4.18(t, J=7.4 Hz, 2H), 4.07 (bs, 2H), 2.90 (dt, J=7.4, 1.9 Hz, 2H), 2.07 (s,3H). Mass spectrum (LCMS, ESI) calc'd for C₁₈H₁₈F₃N₂O₃ (M+H): 367.1.Found: 367.1.

11.N-[4-Amino-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-phenylacetamide

To a solution of2-[2-amino-5-(2,2-difluoro-2-phenylacetylamino)-6-fluorophenyl] ethylacetate (0.67 g, 1.84 mmol), as prepared in the preceding step, in MeOH(19 mL) was added dropwise a solution of K₂CO₃ (280 mg, 2.03 mmol) inH₂O (4.8 mL). The mixture was stirred for 45 minutes, and thenneutralized with 1N HCl. The MeOH was evaporated, and the mixture wasextracted with EtOAc twice. The extracts were combined, washed with H₂O,dried over Na₂SO₄, and concentrated to give the title compound (0.55 g,92% yield) as a pale yellow solid. ¹H-NMR (400 MHz, CD₃OD) δ 7.70-7.68(m, 2H), 7.54-7.48 (m, 3H), 7.01 (t, J=8.5 Hz, 1H), 6.53 (dd, J=8.6, 1.3Hz, 1H), 3.70 (t, J=6.7 Hz, 2H), 2.80 (dt, J=6.7, 2.0 Hz, 2H). Massspectrum (LCMS, ESI) calc'd for C₁₆H₁₆F₃N₂O₂ (M+H): 325.1. Found: 325.3.

12.N-[4-Chloro-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-phenylacetamide(Yokomoto, M, W., et al. 1991, EP 0470 578 A1).

A suspension ofN-[4-amino-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-phenylacetamide(1.63 g, 5.00 mmol), as prepared according to the procedure of thepreceding step, in 6N HCl (9 mL) was cooled in an ice-bath, and then asolution of NaNO₂ (434 mg, 6.30 mmol) in H₂O (2.4 mL) was added over aperiod of 5 minutes. After 30 minutes, acetic acid (2.9 mL) andconcentrated HCl (2.9 mL) were added and the reaction mixture stirredfor 1 hour. To this mixture was added a solution of CuCl (848 mg, 8.55mmol) in concentrated HCl (5 mL) over a period of 20 minutes. Afterstirring in an ice-bath for 3 hours, the reaction mixture was extractedwith EtOAc (200 mL×3). The extracts were combined, washed with H₂O (2times) and brine, dried over Na₂SO₄, concentrated, and flashchromatographed on silica gel eluting with EtOAc/DCM (0, 2.5, and 5%) todeliver the title compound (0.845 g, 48% yield) as a pale orange oil.¹H-NMR (400 MHz, CDCl₃)δ 8.31 (s, 1H), 8.14 (t, J=8.6 Hz, 1H), 7.68-7.66(m, 2H), 7.54-7.46 (m, 3H), 7.19 (dd, J=8.9, 1.7 Hz, 1H), 3.86 (dd,J=12.6, 6.5 Hz., 2H), 3.09 (dt, J=6.7, 2.3 Hz, 2H), 1.58 (t, J=5.7 Hz,1H). Mass spectrum (LCMS, ESI) calc'd for C₁₆H₁₄ClF₃NO₂ (M+H): 344.1.Found: 344.2.

13.2-{3-[(2,2-Difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}ethanol

To a solutionN-[4-chloro-2-fluor-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-phenylacetamide(1.05 g, 3.06 mmol), as prepared according to the procedure in thepreceding step, in THF (12 mL) at 0° C. under argon was added a solutionof borane-THF complex in THF (12.3 mL, 12.3 mmol, 1.0 M) over a periodof 10 minutes, and the reaction mixture continued to stir until theice-bath expired. The reaction mixture was heated at reflux for 20hours, and allowed to cool to room temperature. A solution of K₂CO₃ (1.7g, 12 mmol) in H₂O (12 mL) was added, the THF was removed in vacuo, andthe mixture was extracted with DCM (3 times). The extracts werecombined, washed with brine, dried over Na₂SO₄, concentrated, and flashchromatographed on silica gel eluting with EtOAC/DCM (0 and 2.5%) togive the title compound (815 mg, 81% yield) as a colorless oil. ¹H-NMR(400 MHz, CDCl₃) δ 7.52-7.43 (m, 5H), 6.97 (dd, J=8.8, 1.7 Hz, 1H), 6.51(t, J=8.9 Hz, 1H), 4.17 (bs, 1H), 3.83 (t, J=6.9 Hz, 2H), 3.74 (dt,J=13.4, 6.6 Hz, 2H), 3.04 (dt, J=6.9, 2.4 Hz, 2H), 1.43 (s, 1H). Massspectrum (LCMS, ESI) calc'd for C₁₆H₁₆ClF₃NO (M+H): 330.1. Found: 330.3.

14.2-{3-[(2,2-Difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}ethanal

To a solution of DMSO (1.03 mL, 14.5 mmol), DIEA (1.99 mL, 11.4 mmol),and2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}ethanol(1.45 g, 4.4 mmol), as prepared according to the procedure in thepreceding step, in DCM (140 mL) in an ice-bath was added sulfur trioxidepyridine complex (1.82 g, 11.4 mmol) and stirred at the same temperaturefor 3.5 hours. The mixture was diluted with DCM (300 mL), washed with10% citric acid (3 times), H₂O, and brine, dried over Na₂SO₄, andconcentrated to give the title compound (1.43 g, 99% yield) as an orangeoil, that was used without further purification.

15.2-{3-[(2,2-Difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}aceticAcid (Dalcanale, E., et al. J. Org. Chem., 51:567 (1986)).

A solution of sodium chlorite (692 mg, 6.11 mmol) in H₂O (6.1 mL) wasadded over a period of 30 minutes to a stirred mixture of2{-3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}ethanal(1.43 g, 4.37 mmol), as prepared in the preceding step, in DMSO (4.5 mL)and of NaH₂PO₄ (141 mg, 1.18 mmol) in H₂O (1.7 mL). After the addition,the mixture was stirred at ambient temperature overnight, acidified with10 M HCl to pH 1, and extracted with DCM (3 times). The extracts werecombined, washed with H₂O and brine, dried over Na₂SO₄, and concentratedin vacuo. The resulting residue was flash chromatographed on silica geleluting with MeOH/DCM (0, 2, and 4%) to give the title compound (0.77 g,51% yield) as a pale brown solid. ¹H-NMR (400 MHz, CD₃OD) δ 7.53-7.51(m, 2H), 7.44-7.41 (m, 3H), 6.93 (dd, J=8.9, 1.8 Hz, 1H), 6.62 (t, J=9.1Hz, 1H), 3.80 (t, J=13.7 Hz, 2H), 3.74 (d, J=2.2 Hz, 2H). Mass spectrum(LCMS, ESI) calc'd for C₁₆H₁₄ClF₃NO₂ (M+H): 344.1. Found: 344.4.

16. tert-Butyl2-aza-3-{[2-(2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetylamino)ethoxy]amino}-3-[(tert-butoxy)carbonylamino]prop-2-enate

To a solution of2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}aceticacid (28 mg, 82 μmol), as prepared in the preceding step, in DMF (0.3mL) in an ice-bath was added BOP (58 mg, 130 μmol), HCl salt of[N,N′-di(tert-butoxycarbonyl)]-2-aminoethoxyguanidine (36 mg, 102 μmol)(Tianbao Lu, et al., WO 99/26926 (1999)), and a solution of DIEA (42 mg,33 μmol) in DMF (0.1 mL). After the ice-bath expired, the reactionmixture continued to stir at ambient temperature overnight. The solventswere evaporated, and the resulting residue was partitioned betweensaturated NaHCO₃ and DCM. The aqueous phase was extracted with DCM, andthe organic layers were combined, washed with 10% KHSO₄, H₂O, and brine,dried over Na₂SO₄, concentrated, and flash chromatographed on silica geleluting with MeOH/DCM (1%) to give the title compound (44 mg, 83% yield)as a colorless oil. ¹H-NMR (400 MHz, CDCl₃) δ 7.54-7.50 (m, 2H),7.45-7.42 (m, 3H), 6.93 (dd, J=8.8, 1.7 Hz, 1H), 6.63 (t, J=9.0 Hz, 1H),4.03 (t, J=4.8 Hz, 2H), 3.81 (t, J=13.7 Hz, 2H), 3.71 (d, J=1.9 Hz, 2H),3.47 (t, J=5.1 Hz, 2H), 1.50 (s, 9H), 1.48 (s, 9H). Mass spectrum (LCMS,ESI) calc'd for C₂₉H₃₈ClF₃N₅O₆ (M+H): 644.2. Found: 644.1.

17. N-[2-(Amidinoaminooxy)ethyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetamide Trifluoroacetate Salt

A solution of tert-butyl 2-aza-3-{[2-(2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetylamino)ethoxy]amino}-3-[(tert-butoxy)carbonylamino]prop-2-enoate (44 mg, 68 μmol), as prepared in thepreceding step, in TFA/DCM (2 mL, 2/3) was stirred at room temperaturefor 4 hours. The solvents were evaporated, and the resulting residue wasflash chromatographed on silica gel eluting with 0.05% TFA in MeOH/DCM(5 and 10%) to deliver the title compound (37 mg, 98% yield) as a whitesolid. ¹H-NMR (400 MHz, CD₃OD) δ 7.67-7.52 (m, 2H), 7.47-7.42 (m, 3H),6.96 (dd, J=8.9, 1.7 Hz, 1H), 6.66 (t, J=9.1 Hz, 1H), 3.93 (t, J=5.4 Hz,2H), 3.82 (t, J=13.8 Hz, 2H), 3.71 (d, J=2.0 Hz, 2H), 3.50 (t, J=5.4 Hz,2H). Mass spectrum (LCMS, ESI) calc'd for C₁₉H₂₂ClF₃N₅O₂ (M+H): 444.1.Found: 444.2.

Example 2N-[6-Amino-2-methyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetamideHydrochloride Salt

1.2-{3-[(2,2-Difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}-N-({6-[(tert-butoxy)carbonylamino]-2-methyl(3-pyridyl)}methyl)acetamide

To a solution of2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}aceticacid (516 mg, 1.5 mmol), as prepared in step 15 of Example 1, in DMF(8.0 mL) was added N-[5-(aminomethyl)-6-methyl (2-pyridyl)](tert-butoxy)carboxamide (498 mg, 2.1 mmol) (Sanderson, P. E., et al., WO 97/01338(1997)), BOP (1.06 g, 2.4 mmol), and DIEA (0.78 mL, 4.5 mmol). Afterstirring for 18 hours, additional amine (107 mg, 450 μmol) was added,and the mixture continued to stir for 18 hours. The solvents wereevaporated, and the reaction mixture was partitioned between DCM andsaturated NaHCO₃. The organic layer was separated, and the aqueous layerextracted with DCM. The organic layers were combined, washed with 10%KHSO₄ (2 times), H₂O, and brine, dried over Na₂SO₄, concentrated, andflash chromatographed on silica gel eluting with MeOH/DCM (0, 1.5, and2.5%) to give the title compound (770 mg, 91% yield) as a pale brownfoam. ¹H-NMR (400 MHz, CDCl₃)δ 7.66 (d, J=8.4 Hz, 1H), 7.51-7.41 (m,6H), 7.23 (bs, 1H), 7.01 (dd, J=8.8, 1.5 Hz, 1H), 6.57 (t, J=9.0 Hz,1H), 5.64 (br s, 1H), 4.37 (d, J=5.6 Hz, 2H), 4.26-4.22 (m, 1H),3.79-3.70 (m, 4H), 2.35 (s, 3H), 1.50 (s, 9H). Mass spectrum (LCMS, ESI)calc'd for C₂₈H₃₁ClF₃N₄O₃ (M+H): 563.2. Found: 562.9.

2.N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetamideHydrochloride Salt

To a flask charged with2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}-N-({6-[(tert-butoxy)carbonylamino]-2-methyl(3-pyridyl)}methyl)acetamide(770 mg, 1.37 mmol), as prepared in the preceding step, was added asolution of HCl in 1,4-dioxane (5 mL, 20 mmol, 4.0 M). After stirring atambient temperature for 1.5 hours, some solid precipitated. A solutionof MeOH (1 mL) in DCM (3 mL) was added to dissolve the solid, and themixture was stirred for additional 4 hours. The solvents were removed,and the resulting residue was washed with DCM (5 mL×2), ether (8 mL×2),and dried in high vacuum to give the title compound (620 mg, 91% yield)as a pale brown solid. ¹H-NMR (400 MHz, CD₃OD) δ 7.81 (d, J=9.1 Hz, 1H),7.54-7.51 (m, 2H), 7.45-7.42 (m, 3H), 6.94 (dd, J=8.9, 1.6 Hz, 1H), 6.80(d, J=9.1 Hz, 1H), 6.65 (t, J=9.1 Hz, 1H), 4.25 (s, 2H), 3.81 (t, J=13.8Hz, 2H), 3.70 (d, J=1.8 Hz, 2H), 2.50 (s, 3H). Mass spectrum (LCMS, ESI)calcd for C₂₃H₂₃ClF₃N₄O (M+H): 463.1. Found: 463.7.

Example 3N-[(6-Amino-2,4-dimethyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetamideHydrochloride Salt

1.2-{3-[(2,2-Difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}-N-({6-[(tert-butoxy)carbonylamino]-2,4-dimethyl(3-pyridyl)}methyl)acetamide

To a solution of2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}aceticacid (25 mg, 73 μmol), as prepared in step 15 of Example 1, in DMF (0.25mL) was added BOP (52 mg, 116 μmol), a solution of DIEA (38 mg, 295 mol)in DMF (0.1 mL), andN-[5-(aminomethyl)-4,6-dimethyl(2-pyridyl)](tert-butoxy)carboxamide (23mg, 91 μmol) (Sanderson, P. E., et al. WO 97/01338 (1997)). Afterstirring at ambient temperature for 2 days, additional amine (7 mg, 28μmol) BOP (16 mg, 36 μmol), and DIEA (9 mg, 70 μmol) were added, and themixture was stirred for another 16 hours. The solvents were evaporated,and the resulting residue was partitioned between saturated NaHCO₃ andDCM. The organic layer was separated, and the aqueous layer wasextracted with DCM. The organic layers were combined, washed with 10%citric acid, H₂O, and brine, dried over Na₂SO₄, concentrated, and flashchromatographed on silica gel eluting with MeOH/DCM (0, 1, 2%) toproduce the title compound (18.5 mg, 44% yield) as a pale brown solid.¹H-NMR (400 MHz, CDCl₃) δ 7.58 (s, 1H), 7.50-7.41 (m, 5H), 7.24 (s, 1H),6.98 (dd, J=8.8, 1.1 Hz, 1H), 6.55 (t, J=9.0 Hz, 1H), 5.33 (bs, 1H),4.40 (d, J=4.7 Hz, 2H), 4.24-4.20 (m, 1H), 3.78-3.70 (m, 4H), 2.39 (s,3H), 2.28 (s, 3H), 1.50 (s, 9H). Mass spectrum (LCMS, ESI) calcd forC₂₉H₃₃ClF₃N₄O₃ (M+H): 577.0. Found: 577.1.

2.N-[(6-Amino-2,4-dimethyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetamideHydrochloride Salt

A solution of HCl in 1,4-dioxane (4.0 M, 0.5 mL, 2 mmol) was added to2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}-N-({6-[(tert-butoxy)carbonylamino]-2,4-dimethyl(3-pyridyl)}methyl) acetamide (18.5 mg, 32μmol), as prepared in the preceding step. After stirring at ambienttemperature for 3 hours, solid precipitated. A solution of MeOH (0.1 mL)in DCM (1 mL) was added to dissolve the solid. After stirring foranother 2 hours the reaction was concentrated to give a brown solid,that was washed with ether and DCM and dried in vacuo to produce thetitle compound (12.6 mg, 77% yield) as a pale brown solid. ¹H-NMR (400MHz, CD₃OD) δ 8.40 (bs, 1H), 7.53-7.51 (m, 2H), 7.47-7.40 (m, 3H), 6.93(dd, J=8.8, 1.3 Hz, 1H), 6.68 (s, 1H), 6.64 (t, J=9.1 Hz, 1H), 4.31 (d,J=4.6 Hz, 2H), 3.81 (t, J=13.8 Hz, 2H), 3.67 (d, J=1.2 Hz, 2H), 2.54 (s,3H), 2.42 (s, 3H). Mass spectrum (LCMS, ESI) calc'd for C₂₄H₂₅ClF₃N₄O(M+H): 477.2. Found: 477.5.

Example 4N-[2-(Amidinoaminooxy)ethyl]-2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)acetamideTrifluoroacetate Salt

1. Ethyl 2-(4-fluoronaphthyl)-2-oxoacetate

A solution of n-butyllithium (2.5 M in THF, 20 mL, 50 mmol) was cooledto −78° C., and a solution of 1-bromo-4-fluoronaphthalene (11.25 g, 50mmol) in THF (40 mL) was added slowly and the mixture was stirred for 1hour. The reaction mixture was warmed to −20° C., then added to asolution of diethyl oxalate (29.2 g, 200 mmol) in THF (40 mL) at −78° C.After slowly warming-up to room temperature, EtOAc (100 mL), 10% HCl (50mL) and water (50 mL) were added and the phases were separated. Theaqueous layer was extracted with EtOAc (2×100 mL), and the organiclayers were combined, washed with brine (50 mL), and dried over Na₂SO₄.After evaporating the solvent and the excess diethyl oxalate under highvacuum, the residue was purified by flash column chromatography (1:1DCM:hexane) to give the title compound (9.4 g, 76% yield) as a whitesolid. ¹H—NMR (400 MHz, CDCl₃) δ (9.13 (d, J=8.6 Hz, 1H), 8.20 (d, J=8.4Hz, 1H), 8.01 (dd, J=8.2, 5.4 Hz, 1H), 7.76 (t, J=7.2 Hz, 1H), 7.67 (t,J=8.1 Hz, 1H), 7.21 (t, J=8.5 Hz, 1H), 4.49 (q, J=7.1 Hz, 2H), 1.45 (t,J=7.1 Hz, 3H).

2. Ethyl 2,2-difluoro-2-(4-fluoronaphthyl)acetate

To a solution of ethyl 2-(4-fluoronaphthyl)-2-oxoacetate (9.4 g, 38.2mmol), as prepared in the preceding step, in DCM (60 mL) was added DAST(16.1 g, 100 mmol). The mixture was stirred at room temperatureovernight, poured into ice slowly, and extracted with DCM (3×50 mL). Theorganic layers were combined, washed with brine, and dried over Na₂SO₄.After evaporating the solvent, the residue was purified by flash columnchromatography (1:1 DCM:hexane) to give the title compound (9.7 g, 95%yield) as a light brown oil. ¹H-NMR (400 MHz, CDCl₃) δ 8.20 (m, 2H),7.82 (dd, J=8.2, 5.3 Hz, 1H), 7.63 (m, 2H), 7.20 (t, J=8.4 Hz, 1H), 4.28(q, J=7.1 Hz, 2H), 1.24 (t, J=7.1 Hz, 3H).

3. 2,2-Difluoro-2-(4-fluoronaphthyl)acetic Acid

To a solution of ethyl 2,2-difluoro-2-(4-fluoronaphthyl)acetate (9.6 g,35.8 mmol), as prepared in the preceding step, in methanol (20 mL) andTHF (20 mL) was added a solution of NaOH (2.0 g, 50 mmol) in water (40mL). The reaction mixture was stirred at room temperature for 2 hours.After evaporating the methanol and THF in vacuo, the aqueous phase wasacidified to pH 2 using 10% HCl, and extracted with DCM (3×50 mL). Theextracts were combined, washed with brine, dried over Na₂SO₄, andconcentrated to give the title compound (8.1 g, 94% yield) as an offwhite solid. ¹H-NMR (400 MHz, CDCl₃) δ 9.68 (br s, 1H), 8.18 (m, 2H),7.83 (dd, J=8.1, 5.3 Hz, 1H), 7.62 (m, 2H), 7.19 (t, J=8.3 Hz, 1H).

4.2-{3-[2,2-Difluoro-2-(4-fluoronaphthyl)acetylamino]-2-fluoro-6-nitrophenyl}ethylAcetate

To a solution of DIEA (7.8 mL) and2-(3-amino-2-fluoro-6-nitrophenyl)ethyl acetate (4.6 g, 19 mmol),prepared as in step 8 of Example 1, in DCM (60 mL) was added2,2-difluoro-2-(4-fluoronaphthyl)acetyl chloride (prepared by refluxing2,2-difluoro-2-(4-fluoronaphthyl)acetic acid, as prepared in thepreceding step, with oxalyl chloride) (7.8 g, 30 mmol) in DCM (40 mL).The mixture was stirred at room temperature for 1 hour. Additional DCM(100 mL) was added, and the resulting mixture was washed with 10% citricacid (3×40 mL) and brine, and dried over Na₂SO₄. After evaporating thesolvent, the residue was purified by flash column chromatography elutingwith DCM to give the title compound (5.3 g, 61%) as a yellow oil. ¹H-NMR(400 MHz, CDCl₃) δ 8.45 (m, 2H), 8.21 (d, J=7.0 Hz, 2H), 7.87 (s, 1H),7.85 (t, J=4.1 Hz, 1H), 7.66 (m, 2H), 7.22 (t, J=8.3 Hz, 1H), 4.34 (t,J=6.4 Hz, 2H), 3.36 (t, J=6.4 Hz, 2H), 2.00 (s, 3H).

5.2-{2-Amino-5-[2,2-difluoro-2-(4-fluoronaphthyl)acetylamino]-6-fluorophenyl}ethylAcetate

A mixture of2-{3-[2,2-difluoro-2-(4-fluoronaphthyl)acetylamino]-2-fluoro-6-nitrophenyl}ethylacetate (4.9 g, 10.5 mmol), as prepared in the preceding step, and Pd/C(10%, 500 mg) in ethanol (50 mL) and THF (50 mL) was stirred underhydrogen for 5 hours. The reaction mixture was filtered through Celite,and washed with THF and MeOH. The filtrate and washings were combined,concentrated in vacuo, and flash chromatographed on silica gel elutingwith EtOAc/DCM (0 to 2%) to produce the title compound (3.8 g, 83%) asan off white solid. ¹H-NMR (400 MHz, CDCl₃) δ 8.27 (d, J=8.2 Hz, 1H),8.19 (d, J=8.6 Hz, 1H), 8.10 (s, 1H), 7.82 (m, 2H), 7.63 (m, 2H), 7.19(t, J=8.6 Hz, 1H), 6.46 (d, J=8.7 Hz, 1H), 4.19 (t, J=7.4 Hz, 2H), 4.08(s, 2H), 2.91 (t, J=7.3 Hz, 2H), 2.08 (s, 3H).

6.N-[4-Amino-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-(4-fluoronaphthyl)acetamide

To a solution of2-{2-amino-5-[2,2-difluoro-2-(4-fluoronaphthyl)acetylamino]-6-fluorophenyl}ethylacetate (3.8 g, 8.8 mmol), as prepared in the preceding step, in MeOH(40 mL) and THF (20 mL) was added a solution of K₂CO₃ (1.68 g, 12 mmol)in water (30 mL). The mixture was stirred at room temperature for 3hours. Additional water (50 mL) was added, and the resulting mixture wasextracted with EtOAc (3×50 mL). The extracts were combined, washed withbrine, dried over Na₂SO₄, and concentrated in vacuo to give the titlecompound (3.3 g, 96%) as an off white solid. ¹H-NMR (400 MHz, CDCl₃) δ8.27 (d, J=8.4 Hz, 1H), 8.19 (d, J=8.2 Hz, 1H), 8.13 (s, 1H), 7.81 (m,2H), 7.64 (m, 2H), 7.19 (t, J=8.7 Hz, 1H), 6.47 (d, J=8.7 Hz, 1H), 4.07(s, 2H), 3.89 (t, J=5.6 Hz, 2H), 2.85 (t, J=5.5 Hz, 2H).

7.N-[4-Chloro-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-(4-fluoronaphthyl)acetamide(Doyle, M. P., et al. J. Org. Chem., 42:2426 (1977))

To a flask charged with copper(II) chloride (1.84 g, 13.7 mmol) wasadded a solution of tert-butylnitrite (1.46 g, 12.8 mmol, 90%, Aldrich)in acetonitrile (35 mL) under argon atmosphere. The resulting greenreaction mixture was cooled in an ice-bath to 0° C., and a solution ofN-[4-amino-2-fluor-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-(4-fluoronaphthyl)acetamide (3.58 g, 9.13 mmol), as prepared according to the procedure ofthe preceding step, in acetonitrile (60 mL) was added over a period of45 minutes. After stirring for an additional 6 hours at 0° C., theresulting brown mixture was allowed to warm up to ambient temperature,then poured into 20% aqueous HCl (160 mL), and extracted with DCM (3times). The extracts were combined, washed with 20% HCl, H₂O, and brine,dried over Na₂SO₄, concentrated, and flash chromatographed on silica geleluting with EtOAc/DCM (0 and 2.5%) to deliver the title compound (2.1g, 56% yield) as a white solid. ¹H-NMR (400 MHz, CDCl₃) δ 8.31 (br s,1H), 8.24-8.12 (m, 3H), 7.84 (dd, J=8.2, 5.3 Hz, 1H). 7.68-7.60 (m, 2H),7.22-7.20 (m, 2H), 3.87 (dd, J=12.6, 6.5 Hz, 2H), 3.09 (dt, J=6.7, 2.2Hz, 2H), 1.47 (t, J=5.6 Hz, 2H). Mass spectrum (LCMS, ESI) calc'd forC₂₀H₁₅ClF₄NO₂ (M+H): 412.1. Found: 412.6.

8.2-(3-{[2,2-Difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)ethanol

To a solution ofN-[4-chloro-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-(4-fluoronaphthyl)acetamide(1.9 g, 4.6 mmol), as prepared in the preceding step, in THF (19 mL) at0° C. was added dropwise a solution of BH₃.THF complex (19.4 mL, 19.4mmol, 1.0 M in THF) over a period of 20 minutes, and the reactionmixture continued to stir until the ice bath expired. The mixture wasthen heated at reflux in an oil bath at 75 to 80° C. for 3 hours, andcontinued to stir at ambient temperature overnight. A solution of NaHCO₃(1.63 g, 19.4 mmol) in H₂O (20 mL) was added, the THF was evaporated,and the resulting mixture was extracted with DCM twice. The extractswere combined, washed with H₂O and brine, dried over Na₂SO₄,concentrated, and flash chromatographed on silica gel eluting withEtOAc/DCM (0, 1, 1.5%) to give the title compound (805 mg, 44% yield) asa white solid. ¹H-NMR (400 MHz, CDCl₃) δ 8.24-8.19 (m, 2H), 7.70-7.61(m, 3H), 7.14 (t, J=9.3 Hz, 1H), 6.90 (dd, J=8.7, 1.6 Hz, 1H), 6.42 (t,J=8.9 Hz, 1H), 4.22-4.16 (m, 1H), 4.00 (dt, J=13.4, 6.8 Hz, 2H), 3.82(dd, J=12.1, 6.4 Hz, 2H), 3.02 (dt, J=6.8, 2.3 Hz, 2H), 1.39 (br s, 1H).Mass spectrum (LCMS, ESI) calc'd for C₂₀H₁₇ClF₄NO (M+H): 398.1. Found:398.3.

9.2-(3-{[2,2-Difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)ethanal

To a solution of DMSO (470 mg, 6.0 mmol), DIEA (823 μL, 4.74 mmol) and2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)ethanol(723 mg, 1.82 mmol), as prepared in the preceding step, in DCM (55 mL)in an ice-bath was added sulfur trioxide pyridine complex (754 mg, 4.74mmol). After stirring for 3.5 hours the reaction mixture was dilutedwith DCM (110 mL). The organic layer was separated, and the aqueouslayer extracted with DCM (100 mL). The organic layers were combined,washed with 10% citric acid (3 times), H₂O, and brine, dried overNa₂SO₄, and concentrated to give the title compound (722 mg,quantitative yield) as an orange oil. ¹H-NMR (400 MHz, CDCl₃) δ 9.69 (d,J=1.1 Hz, 1H), 8.22-8.18 (m, 2H), 7.67-7.60 (m, 3H), 7.14 (t, J=9.2 Hz,1H), 6.94 (d, J=8.8 Hz, 1H), 6.49 (t, J=8.7 Hz, 1H), 4.20 (bs, 1H), 4.00(dt, J=13.3, 6.7 Hz, 2H), 3.83 (s, 2H).

10.2-(3-{[2,2-Difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)aceticAcid

A solution of sodium chlorite (309 mg, 2.73 mmol, 80%) in H₂O (3.0 mL)was added dropwise over a period of 30 minutes to a stirred mixture of2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)ethanal(722 mg, 1.82 mmol), as prepared in the preceding step, in DMSO (3.6 mL)and of NaH₂PO₄ (74 mg, 0.55 mmol) in H₂O (0.9 mL). After the addition,the mixture was stirred at ambient temperature for 48 hours, thenacidified with 10 M HCl to pH 1, and extracted with DCM (3 times). Theextracts were combined, washed with H₂O and brine, dried over Na₂SO₄,and concentrated in vacuo. The resulting residue was flashchromatographed on silica gel eluting with MeOH/DCM (0, 1, 1.5, and 2%)to give the starting aldehyde2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)ethanal(165 mg, 23% yield) and the title compound (570 mg, 76% yield) as asolid. ¹H-NMR (400 MHz, CDCl₃) δ 8.32 (d, J=8.4 Hz, 1H), 8.15 (d, J=8.0Hz, 1H), 7.71-7.61 (m, 3H), 7.18 (dd, J=9.8, 8.5 Hz, 1H), 6.78 (dd,J=8.8, 1.5 Hz, 1H), 6.44 (t, J=9.1 Hz, 1H), 4.05 (t, J=13.5 Hz, 2H),3.69 (d, J=2.1 Hz, 2H).

11. tert-Butyl2-aza-3-({2-[2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)acetylamino]ethoxy}amino)-3-[(tert-butoxy)carbonylamino]prop-2-enoate

To a solution of2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)aceticacid (570 mg, 1.39 mmol), as prepared in the preceding step, in DMF (7.5mL) in an ice-bath was added BOP (981 mg, 2.22 mmol), HCl salt of[N,N′-di(tert-butoxycarbonyl)]-2-aminoethoxyguanidine (689 mg, 1.94mmol), and DIEA (0.96 mL, 5.55 mmol). After the ice-bath expired, themixture continued to stir at ambient temperature overnight. AdditionalBOP (123 mg, 0.28 mmol) and the HCl salt of[N,N′-di(tert-butoxycarbonyl)]-2-aminoethoxyguanidine (98 mg, 0.28 mmol)were added, and the reaction mixture was stirred for 24 hours. Thesolvents were evaporated, and the resulting residue was partitionedbetween saturated NaHCO₃ and DCM. The organic layer was separated, andthe aqueous layer was extracted with DCM. The organic layers werecombined, washed with 10% KHSO₄, H₂O, and brine, dried over Na₂SO₄,concentrated, and flash chromatographed on silica gel eluting withMeOH/DCM (0, 0.5, and 1%) to give the title compound (720 mg, 73% yield)as a white foam. ¹H-NMR (400 MHz, CD₃OD) δ 9.12 (s, 1H), 8.24-8.19 (m,2H), 7.78-7.81 (m, 1H), 7.70-7.60 (m, 4H), 7.14 (dd, J=9.6, 8.4 Hz, 1H),6.91 (dd, J=8.8, 1.6 Hz, 1H), 6.44 (t, J=8.8 Hz, 1H), 4.20-4.15 (m, 1H),4.13-4 10 (m, 2H), 3.98 (dt, J=13.4, 6.7 Hz, 2H), 3.78 (d, J=1.9 Hz,2H), 3.60 (dd, J=8.6, 4.9 Hz, 2H), 1.51 (s, 9H), 1.48 (s, 9H). Massspectrum (LCMS, ESI) calc'd for C₃₃H₃₉ClF₄N₅O₆ (M+H): 712.2. Found:712.3.

12.N-[2-(Amidinoaminooxy)ethyl]-2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)acetamideTrifluoroacetate Salt

A solution of tert-butyl 2-aza-3-({2-[2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)acetylamino]ethoxy}amino)-3-[(tert-butoxy)carbonylamino]prop-2-enoate (720 mg,1.01 mmol), as prepared in the preceding step, in TFA/DCM (2:3, 30 mL)was stirred at ambient temperature for 4 hours. The solvents wereevaporated, and the resulting residue was flash chromatographed onsilica gel eluting with 0.05% TFA in MeOH/DCM (5 and 10%) to give thetitle compound (626 mg, 99% yield) as a pale brown foam. ¹H-NMR (400MHz, CD₃OD) δ 8.33 (d, J=8.5 Hz, 1H), 8.17-8.15 (m, 1H), 7.73-7.63 (m,3H), 7.20 (dd, J=10.0, 8.3 Hz, 1H), 6.82 (dd, J=8.8, 1.6 Hz, 1H), 6.92(t, J=9.1 Hz, H), 4.07 (t, J=13.7 Hz, 2H), 3.92 (t, J=5.4 Hz, 2H), 3.67(d, J=1.8 Hz, 2H), 3.52-3.51 (m, 2H). Mass spectrum (LCMS, ESI) calc'dfor C₂₃H₂₃ClF₄N₅O₂ (M+H): 512.1. Found: 512.2.

Example 5N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)acetamideHydrochloride Salt

1.2-(3-{[2,2-Difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)-N-({6-[(tert-butoxy)carbonylamino]-2-methyl(3-pyridyl)}methyl)acetamide

To a solution of2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)aceticacid (15 mg, 37 μmol), as prepared according to the procedure of step 10of Example 4, in DMF (0.3 mL) was added BOP (26 mg, 58 μmol),N-[5-(aminomethyl)-6-methyl(2-pyridyl)](tert-butoxy)carboxamide (12 mg,51 μmol), and a solution of DIEA (19 mg, 146 μmol) in DMF (0.1 mL)(Sanderson, P. E., et al., WO 97/01338 (1997)). The mixture was stirredovernight, the solvents evaporated, and the resulting mixture waspartitioned between saturated NaHCO₃ and DCM. The organic layer wasseparated, and the aqueous layer extracted with DCM. The organic layerswere combined, washed with 10% KHSO₄, H₂O, and brine, dried over Na₂SO₄,concentrated, and flash chromatographed on silica gel eluting withMeOH/DCM (0.3, 0.6, and 1%) to give the title compound (11 mg, 49%yield) as a white solid. ¹H-NMR (400 MHz, CD₃OD) δ 8.33 (d, J=8.4 Hz,1H), 8.17-8.14 (m, 1H), 7.72-7.61 (m, 4H), 7.55 (d, J=8.5 Hz, 1H), 7.19(dd, J=10.0, 8.3 Hz, 1H), 6.81 (dd, J=8.8, 1.7 Hz, 1H), 6.47 (t, J=9.1Hz, 1H), 4.32 (s, 2H), 4.12-4.03 (m, 2H), 3.67 (d, J=2.0 Hz, 2H), 2.40(s, 3H), 1.50 (s, 9H). Mass spectrum (LCMS, ESI) calc'd forC₃₂H₃₂ClF₄N₄O₃ (M+H): 631.2. Found: 631.1.

2.N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(3-{[(2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)acetamideHydrochloride Salt

A solution of2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)-N-({6-[(tert-butoxy)carbonylamino]-2-methyl(3-pyridyl)}methyl)acetamide(10 mg, 16 μmol), as prepared in the preceding step, in HCl (0.5 mL, 4.0M in 1,4-dioxane) was stirred for 2 hours at ambient temperature. Asolution of MeOH/DCM (25%, 0.4 mL) was added, and the mixture continuedto stir overnight. The solvents were evaporated, and the resulting brownresidue was flash chromatographed on silica gel eluting with MeOH/DCM(2.5, 5, and 10%) to give a solid product. It was treated with HClsolution (0.01 mL, 4.0 M in 1,4-dioxane, 40 μmol) in DCM (0.5 mL),stirred for 5 minutes, and the solvents evaporated to give the titlecompound (6.2 mg, 69% yield) as a white solid. ¹H-NMR (400 MHz, CD₃OD) δ8.26 (d, J=8.4 Hz, 1H), 8.17-8.14 (m, 1H), 7.80 (d, J=9.1 Hz, 1H),7.73-7.64 (m, 4H), 7.20 (dd, J=10.0, 8.3 Hz, 1H), 6.82-6.79 (m, 1H),6.49 (t, J=9.1 Hz, 1H), 4.24 (s, 2H), 4.10-4.02 (m, 2H), 3.66 (d, J=2.3Hz, 2H), 2.49 (s, 3H). Mass spectrum (LCMS, ESI) calc'd forC₂₇H₂₄ClF₄N₄O (M+H): 531.1. Found: 531.6.

Example 6N-[2-(Guanidinooxy)ethyl]-2-(3-{[benzylsulfonyl]amino}phenyl)acetamideTrifluoroacetate Salt

1. N-(2-Hydroxyethyl)-2-(3-nitrophenyl)acetamide

To a solution of 3-nitrophenylacetic acid (3.21 g, 17.7 mmol),ethanolamine (2.8 g, 46 mmol), and triethylamine (3.0 mL, 22 mmol) inanhydrous DMF (110 mL) was added a solution ofbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP, 9.37 g, 18.0 mmol) in anhydrous DMF (80 mL). After stirring 16hours at ambient temperature (under nitrogen), the reaction mixture wasconcentrated in vacuo, dissolved in DCM and filtered. The filtrate waswashed with 10% aqueous citric acid, saturated aqueous NaHCO₃, pH 7buffer, and brine, dried over Na₂SO₄ and filtered. The evaporatedfiltrate was then purified by flash chromatography (10% methanol in DCM)giving the title compound (1.02 g, 26%) as a light yellow solid. ¹H NMR(300 MHz, CDCl₃/CD₃OD) δ 8.18 (m, 1H), 8.11 (ddd, 1H, J=8.1 Hz, 2.4 Hz,1.1 Hz), 7.72 (m, 1H), 7.60 (t, 1H, J=7.8 Hz), 3.62 (s, 2H), 3.44 (t,2H, J=5.9 Hz), 3.16 (t, 2H, J=5.9 Hz).

2. N-[2-(N′-Phthalimidyl)hydroxyethyl]-2-(3-nitrophenyl)acetamide

To a solution of the product of the preceding step (1.02 g, 4.55 mmol),N-hydroxyphthalimide (0.76 g, 4.64 mmol), and triphenylphosphine (1.22g, 4.65 mmol) in anhydrous THF (100 mL) was addeddiethylazadicarboxylate (0.75 mL, 4.77 mmol) via syringe. After stirringovernight at ambient temperature (under nitrogen), the reaction wasconcentrated in vacuo and purified by flash chromatography (40% ethylacetate in DCM) giving an impure product that was dissolved in DCM,cooled, and filtered. The evaporated filtrate was then purified by flashchromatography (66%-100% ethyl acetate in hexane) giving the titlecompound (0.86 g, 51%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.26(t, 1H, J=1.7 Hz), 8.15 (ddd, 1H, J=8.3 Hz, 2.3 Hz, t, 1.0 Hz), 7.82 (m,4H), 7.74 (m, 1H), 7.53 (t, 1H, J=7.9 Hz), 7.03 (br s, 1H), 4.26 (m,2H), 3.76 (s, 2H), 3.57 (dd, 2H, J=9.8 Hz, 5.7 Hz).

3. N-[2-(N′-Phthalimidyl)hydroxyethyl]-2-(3-aminophenyl)acetamide

A solution of the product of the preceding step (0.66 g, 1.80 mmol) and10% palladium on carbon (15 mg) in degassed 1:1 ethanol:THF (40 mL) wasstirred under hydrogen at ambient temperature. After 6 hours thereaction was filtered over Celite and the filtrate evaporated andpurified by flash chromatography (5% methanol in DCM) giving the titlecompound (0.20 g, 33%) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 7.81(m, 4H), 7.14 (t, 1H, J=8.0 Hz), 6.72 (m, 2H), 6.61 (ddd, 1H, J=8.0 Hz,2.2 Hz, 1.0 Hz), 4.23 (m, 2H), 3.55 (m, 4H). Mass spectrum (MALDI-TOF,α-cyano-4-hydroxycinnamic acid matrix) calc'd. for C₁₈H₁₇N₃O₄: 362.1(M+Na), 340.1 (M+H). Found: 362.2, 340.3.

4.N-[2-(N′-Phthalimidyl)hydroxyethyl]-2-(3-{[benzylsulfonyl]-amino}phenyl)acetamide

To an ice-cold solution of the product of the preceding step (0.20 g,0.58 mmol) in anhydrous DCM (50 mL) was added a solution ofα-toluenesulfonyl chloride (0.11 g, 0.58 mmol) in anhydrous DCM (20 mL)followed by N-methylmorpholine (0.10 mL, 0.91 mmol). After stirring 16hours at ambient temperature, more α-toluenesulfonyl chloride (0.07 g,0.36 mmol) and N-methylmorpholine (0.10 mL, 0.91 mmol) were added andthe reaction stirred an additional 4 hours and evaporated in vacuo. Theresidue was dissolved in DCM, washed with 10% aqueous citric acid, pH 7buffer and brine, dried over Na₂SO₄, filtered, and the filtrateevaporated giving the title compound (0.20 g, 69%) as a light yellowsolid. ¹H NMR (300 MHz, CDCl₃) δ 7.77 (m, 4H), 7.35 (m, 1H), 7.22 (s,5H), 7.17 (m, 1H), 7.11 (m, 1H), 7.05 (s, 1H), 6.71 (br m, 1H), 4.37 (s,2H), 4.25 (m, 2H), 3.64 (s, 2H), 3.64 (dd, 2H, J=10 Hz, 5.5 Hz).

5. N-[2-(Aminooxy)ethyl]-2-(3-{[benzylsulfonyl]amino}phenyl)acetamide

The product of the preceding step (0.19 g, 0.39 mmol) was dissolved in1:1 ethanol:THF (20 mL) and reacted with 40% aqueous methylamine (10 mL)for 1 hour at ambient temperature. The reaction was evaporated in vacuoand purified on a Waters Sep-Pak (5 g silica, 1:1 DCM:ethyl acetate)giving an impure yellow solid. This was then purified by preparativethin layer chromatography (10% methanol in DCM) giving the titlecompound (0.10 g, 72%) as a white solid. ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ7.34 (m, 3H), 7.28 (m, 3H), 7.10 (m, 1H), 7.05 (m, 2H), 4.35 (s, 2H),3.69 (1, 2H, J=5 Hz), 3.51 (s, 2H), 3.43 (t, 2H, J=5 Hz). Mass spectrum(MALDI-TOF, α-cyano-4-hydroxycinnamic acid matrix) calc'd. forC₁₇H₂₁N₃O₄S: 386.1 (M+Na). Found: 386.6.

6.N-[2-({N,N′-Di[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(3-{[benzylsulfonyl]amino}phenyl)acetamide

A solution of the product of the preceding step (89 mg, 0.24 mmol) and[N,N′-di(tert-butoxycarbonyl)]amidinopyrazole (86 mg, 0.28 mmol) in DMF(5 mL) was stirred for 4 days at ambient temperature. The reaction wasevaporated in vacuo and the crude product purified by flashchromatography (5% methanol in DCM) giving an impure yellow oil. Thiswas then purified by preparative thin layer chromatography (5% methanolin DCM) giving the title compound (72 mg, 49%) as a colorless solid. ¹HNMR (300 MHz, CDCl₃) δ 9.19 (s, 1H), 8.22 (br t, 1H, J=5.0 Hz), 7.62 (s,1H), 7.23 (m, 10H (Ar+NH)), 4.27 (s, 2H), 4.08 (m, 2H), 3.57 (m, 4H),1.51 (s, 9H), 1.49 (s, 9H).

7. N-[2-(Guanidinooxy)ethyl]-2-(3-{[benzylsulfonyl]amino}phenyl)acetamide Trifluoroacetate Salt

The product of the preceding step (72 mg, 0.12 mmol) was dissolved inDCM (5 mL) and reacted with trifluoroacetic acid (2 mL) for 4 hours atambient temperature. The reaction was concentrated in vacuo and thecrude product purified by preparative thin layer chromatography (20%methanol in DCM) giving the title compound (44 mg, 71%) as a pale yellowwax. ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ 7.34 (m, 3H), 7.28 (m, 3H), 7.11(m, 1H), 7.05 (m, 2H), 4.35 (s, 2H), 3.90 (t, 2H, J=4.9 Hz), 3.52 (s,2H), 3.47 (t, 2H, J=4.8 Hz). Mass spectrum (MALDI-TOF,α-cyano-4-hydroxycinnamic acid matrix) calc'd. for C₁₈H₂₃N₅O₄S: 428.1(M+Na), 406.2 (M+H). Found: 428.4, 406.4.

Example 7N-[2-(Guanidinooxy)ethyl]-2-(2-chloro-5-{[benzylsulfonyl]amino}phenyl)acetamide Trifluoroacetate Salt

1. 2-Chloro-5-nitrophenylacetic Acid Monohydrate

A solution of 2-chlorophenylacetic acid (10.0 g, 58.6 mmol) inconcentrated sulfuric acid (40 mL) was cooled to −10° C. and slowlyreacted with a solution of fuming nitric acid (2.80 mL, 66.7 mmol) inconcentrated sulfuric acid (7.2 mL). After 2.5 hours the reaction wasslowly poured over ice water (400 mL), filtered over a coarse filterfrit, washed once with cold water, and dried on the frit overnightgiving the title compound (13.5 g, 98%) as a white solid. Integration ofthe proton NMR spectrum showed the product contained about 0.2equivalents of 2-chlorophenylacetic acid, but thin-layer chromatographyshowed this to be inseparable from the product. ¹H NMR (300 MHz,CDCl₃/CD₃OD) δ 8.25 (d, 1H, J=2.7 z), 8.13 (dd, 1H, J=8.7 Hz, 2.7 Hz),7.61 (d, 1H, J=8.8 Hz), 3.89 (s, 2H).

2. Ethyl 2-(3-amino-6-chlorophenyl)acetate

The product of the preceding step (4.14 g, 17.7 mmol) was suspended inDCM (70 mL) and reacted with oxalyl chloride (4.0 mL, 46 mmol) and a fewdrops of DMF. After stirring 1 hour at ambient temperature the reactionbecame homogeneous, reagent grade ethanol (30 mL) was added and thereaction stirred another 30 minutes. The crude product was evaporated invacuo and purified by flash chromatography (10% to 15% ethyl acetate inhexane) giving the title compound (4.6 g) as a pale yellow oil. ProtonNMR showed the product contained about 0.8 equivalents of diethyloxalatethat could not be located by thin-layer chromatography. ¹H NMR (300 MHz,CDCl₃) δ 8.21 (d, 1H, J=2.7 Hz), 8.11 (dd, 1H, J=8.8 Hz, 2.7 Hz), 7.57(d, 1H, J=8.8 Hz), 4.21 (q, 2H, J=7.2 Hz), 3.87 (s, 2H), 1.28 (t, 3H,J=7.2 Hz).

3. Ethyl 2-(3-amino-6-chlorophenyl)acetate

A solution of the product of the preceding step (2.00 g, 8.21 mmol) inreagent grade ethanol (50 mL) was reacted with tin(II)chloride dihydrate(9.40 g, 41.7 mmol) at ambient temperature. After 16 hours the reactionwas concentrated in vacuo, dissolved in DCM and filtered. The filtratewas washed with water and brine, dried over Na₂SO₄, and filtered. Theevaporated filtrate was then purified by flash chromatography (40% ethylacetate in hexane) giving the title compound (0.53 g, 30%) as a paleyellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.12 (d, 1H, J=8.5 Hz), 6.60 (d,1H, J=2.8 Hz), 6.53 (dd, 1H, 8.5 Hz, 2.9 Hz), 4.17 (q, 2H, J=7.1 Hz),3.65 (s, 2H), 1.26 (t, 3H, J=7.1 Hz).

4. Ethyl 2-(2-chloro-5-{[benzylsulfonyl]amino}phenyl)acetate

A solution of the product of the preceding step (0.50 g, 2.32 mmol) andα-toluenesulfonyl chloride (0.74 g, 3.88 mmol) in DCM (40 mL) andN-methylmorpholine (0.80 mL, 7.3 mmol) was stirred at ambienttemperature for 3 hours, washed with dilute aqueous HCl, water, andbrine, dried over Na₂SO₄, and filtered. The evaporated filtrate waspurified by flash chromatography (5% ethyl acetate in DCM) giving thetitle compound (0.693 g, 81%) as a pale yellow solid. ¹H NMR (300 MHz,CDCl₃) δ 7.36 (m, 4H), 7.26 (m, 2H), 7.04 (d, 1H, J=2.7 Hz), 6.99 (dd,1H, J=8.5 Hz, 2.8 Hz), 6.47 (br s, 1H), 4.33 (s, 2H), 4.20 (q, 2H, J=7.1Hz), 3.73 (s, 2H), 1.29 (t, 3H, J=7.1 Hz). Mass spectrum (MALDI-TOF,α-cyano-4-hydroxycinnamic acid matrix) calc'd. for C₁₇H₁₈NO₄SCl: 390.1(M+Na). Found: 390.7.

5. 2-(2-Chloro-5-{[benzylsulfonyl]amino}phenyl)acetic Acid

A solution of the product of the preceding step (0.69 g, 1.87 mmol) in1:1 water/THF (20 mL) was reacted with potassium hydroxide (0.52 g, 9.32mmol) at ambient temperature for 20 hours. After evaporating the THF invacuo, the remaining aqueous layer was acidified to pH 3 with 1N HCl andextracted with DCM and ether. The combined organic layers were washedwith brine, dried over Na₂SO₄, and filtered. The filtrate was thenevaporated in vacuo to give the title compound (0.586 g, 92%) as a whitesolid. ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ 7.34 (m, 4H), 7.28 (m, 2H), 7.07(m, 2H), 4.34 (s, 2H), 3.73 (s, 2H). Mass spectrum (MALDI-TOF,α-cyano-4-hydroxycinnamic acid matrix) calc'd. for C₁₅H₁₄NO₄SCl: 378.0(M+K), 362.0 (M+Na). Found. 378.8, 362.9.

6.N-[2-({N,N′-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(2-chloro-5-{[benzylsulfonyl]amino}phenyl)acetamide

A solution of the product of the preceding step (0.21 g, 0.60 mmol) and[N,N′-di(tert-butoxycarbonyl)]-2-aminoethoxyguanidine (Tianbao Lu, etal., WO 99/26926 (1999)) (0.19 g, 0.60 mmol), in anhydrous THF (50 mL)was reacted with BOP (0.33 g, 0.75 mmol) and triethylamine (0.25 mL, 1.8mmol) at ambient temperature for 16 hours. The reaction was evaporatedin vacuo, dissolved in DCM, washed with pH 7 buffer and brine, driedover Na₂SO₄, and filtered. The evaporated filtrate was purified by flashchromatography (5% methanol in DCM) giving the title compound (0.380 g,98%) as an orange solid. ¹H NMR (300 MHz, CDCl₃) δ 9.14 (s, 1H), 8.22(br t, 1H, J=5.0 Hz), 7.60 (s, 1H), 7.34 (m, 3H), 7.28 (m, 3H), 7.10 (d,1H, J=2.6 Hz), 7.03 (dd, 1H, J=8.6 Hz, 2.7 Hz), 6.84 (br s, 1H), 4.29(s, 2H), 4.13 (m, 2H), 3.75 (s, 2H), 3.62 (dd, 2H, J=8.8 Hz, 5.1 Hz),1.51 (s, 9H), 1.46 (s, 9H). Mass spectrum (MALDI-TOF, gentisic acidmatrix) calc'd. for C₂₈H₃₈N₅O₈S Cl: 662.2 (M+Na), 440.1 (M−2 Boc+H).Found: 661.7, 439.9.

7.N-[2-(Guanidinooxy)ethyl]-2-(2-chloro-5-{[benzylsulfonyl]-amino}phenyl)acetamideTrifluoroacetate Salt

A solution of the product of the preceding step (0.375 g, 0.586 mmol) inDCM (10 mL) was reacted with trifluoroacetic acid (5 mL) at ambienttemperature for 16 hours. The reaction was evaporated in vacuo, and thecrude product purified by flash chromatography (20% methanol in DCM)giving the title compound (0.326 g, 100%) as a pale yellow solid. ¹H NMR(300 MHz, CDCl₃/CD₃OD) δ 7.34 (m, 3H), 7.29 (m, 3H), 7.06 (m, 2H), 4.35(s, 2H), 3.94 (br t, 2H, J=5 Hz), 3.65 (s, 2H), 3.49 (br t, 2H, J=5 Hz).Mass spectrum (MALDI-TOF, α-cyano-4-hydroxycinnamic acid matrix) calc'd.for C₁₈H₂₂N₅O₄SCl: 462.1 (M+Na), 440.1 (M+H). Found: 461.9, 439.9.

Example 8N-[2-(Guanidinooxy)ethyl]-2-(2-methyl-5-{[benzylsulfonyl]amino}phenyl)acetamideTrifluoroacetate Salt

1. (2-Methyl-5-nitrophenyl)methanol

2-Methyl-5-nitrobenzoic acid (2.00 g, 11.0 mmol) was warmed undernitrogen, dissolved in anhydrous THF (25 mL), and treated with a 1Nsolution of borane in THF (16.5 mL). After stirring 18 hours at ambienttemperature the reaction was quenched with a solution of potassiumcarbonate (1.8 g, 13 mmol) in water (50 mL), and the THF removed invacuo. The remaining aqueous solution was extracted with DCM, and theorganic layer washed with pH 7 buffer and brine, dried over sodiumsulfate, and filtered. The evaporated filtrate gave the title compoundas a pale yellow solid (1.76 g, 95%). ¹H NMR (300 MHz, CDCl₃) δ 8.29 (d,1H, J=2.5 Hz), 8.04 (dd, 1H, J=8.3 Hz, 2.5 Hz), 7.31 (d, 1H, J=8.31 Hz),4.77 (d, 2H, J=5.5 Hz), 2.41 (s, 3H), 2.09 (t, 1H, J 5.6 Hz).

2. (2-Methyl-5-nitrophenyl)methyl Methylsulfonate

A solution of the product of the preceding step (1.74 g, 10.4 mmol) inDCM (50 mL) was cooled to 0° C. and treated with methanesulfonylchloride (0.90 mL, 11.6 mmol) and triethylamine (1.75 mL, 12.6 mmol).After stirring 30 minutes the reaction was warmed to ambienttemperature, stirred another 30 minutes, and poured onto pH 7 buffersolution. The phases were separated and the organic layer washed withbrine, dried over sodium sulfate, and filtered. The evaporated filtrategave the title compound as a pale yellow oil (2.53 g, 99%). ¹H NMR (300MHz, CDCl₃) δ 8.26 (d, 1H, J=2.5 Hz), 8.16 (dd, 1H, J=8.4 Hz, 2.4 Hz),7.42 (d, 1H, J=8.4 Hz), 5.31 (s, 2H), 3.08 (s, 3H), 2.51 (s, 3H).

3. 2-(2-Methyl-5-nitrophenyl)ethanenitrile

The product of the preceding step (2.48 g, 10.1 mmol) and potassiumcyanide (2.00 g, 30.7 mmol) were refluxed in acetonitrile (100 mL) for 8hours, then cooled to ambient temperature and stirred overnight. Thereaction was evaporated in vacuo, dissolved in DCM, and filtered. Thefiltrate was washed with pH 7 buffer and brine, evaporated, and purifiedby flash column chromatography (1:1 hexane:ethyl acetate eluant) givingthe title compound (1.27 g, 71%) as a yellow solid. ¹H NMR (300 MHz,CDCl₃) δ 8.26 (d, 1H, J=2.4 Hz), 8.13 (dd, 1H, J=8.3 Hz, 2.4 Hz), 7.42(d, 1H, J=8.3 Hz), 3.78 (s, 2H), 2.48 (s, 3H).

4. 2-(2-Methyl-5-nitrophenyl)acetic Acid

To a solution of the product of the preceding step (1.27 g, 7.21 mmol)in methanol (30 mL) was added a solution of potassium hydroxide (4.06 g,72.4 mmol) in water (30 mL). The reaction was heated at reflux overnightand the methanol removed in vacuo. The remaining aqueous layer wasacidified with 3N HCl and filtered, the solid washed with diethyl ether,and the filtrate separated. The aqueous layer was washed with DCM anddiethyl ether, and the combined organic layers washed with brine, driedover sodium sulfate, and filtered. The evaporated filtrate gave thetitle compound (0.84 g, 60%) as an orange solid. ¹H NMR (300 MHz,acetone-d₆) δ 8.17 (d, 1H, J=2.3 Hz), 8.06 (dd, 1H, J=8.4 Hz, 2.5 Hz),7.29 (d, 1H, J=8.4 Hz), 3.88 (s, 2H), 2.45 (s, 3H).

5.N-[2-({N,N′-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(2-methyl-5-nitrophenyl)acetamide

A solution of the product of the preceding step (0.27 g, 1.40 mmol), BOP(0.70 g, 1.58 mmol), triethylamine (0.50 mL, 3.60 mmol), and[N,N′-di(tert-butoxycarbonyl)] 2-aminoethoxyguanidine (Tianbao Lu, etal., WO 99/26926 (1999)) (0.44 g, 1.38 mmol), in anhydrous DMF werestirred at ambient temperature overnight. The reaction was concentratedin vacuo and the crude product purified by flash column chromatography(5% methanol in DCM eluant) giving the title compound as a pale orangesolid (0.63 g, 92%). ¹H NMR (300 MHz, CDCl₃) δ 9.21 (s, 1H), 8.42 (m,1H), 8.17 (d, 1H, J=2.4 Hz), 7.99 (dd, 1H, J=8.4 Hz, 2.5 Hz), 7.59 (s,1H), 7.29 (d, 1H, J=8.4 Hz), 4.12 (m, 2H), 3.74 (s, 2H), 3.63 (m, 2H),2.48 (s, 3H), 1.52 (s, 9H), 1.47 (s, 9H).

6.N-[2-({N,N′-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(3-amino-6-methylphenyl)acetamide

The product of the preceding step (0.29 g, 0.58 mmol) and 10% palladium(0) on carbon (0.06 g) were dissolved in reagent ethanol (50 mL),degassed with nitrogen and vacuum, and stirred under a hydrogen balloonat ambient temperature. After 4 hrs the reaction was filtered overCelite, the frit washed with methanol, and the filtrate evaporated invacuo giving the title compound (0.27 g, 100%). ¹H NMR (300 MHz, CDCl₃)δ 9.10 (s, 1H)7.61 (s, 1H), 7.30 (m, 1H), 6.94 (d, 1H, J=8.0 Hz), 6.63(d, 1H, J=2.4 Hz), 6.51 (dd, 1H, J=8.0 Hz, 2.5 Hz), 4.09 (m, 2H), 3.58(m, 2H), 3.53 (s, 2H), 2.21 (s, 3H), 1.52 (s, 9H), 1.47 (s, 9H).

7.N-[2-({N,N′-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(2-methyl-5-{[benzylsulfonyl]amino}phenyl)acetamide

The product of the preceding step (0.27 g, 0.58 mmol), α-toluenesulfonylchloride (0.18 g, 0.96 mmol), and N-methylmorpholine (0.20 mL, 1.82mmol) were stirred at ambient temperature in DCM (20 mL). After 2 hoursthe reaction was diluted with additional DCM and washed with diluteaqueous HCl, saturated aqueous sodium bicarbonate, pH 7 buffer, andbrine. The organic layer was dried over sodium sulfate, filtered, andthe filtrate concentrated in vacuo giving the title compound as a paleyellow solid (0.35 g, 97%). ¹H NMR (300 MHz, CDCl₃) δ 9.15 (s, 1H), 8.04(br t, 1H, J=5 Hz), 7.60 (s, 1H), 7.34 (m, 5H), 7.12 (d, 1H, J=8.0 Hz),7.03 (dd, 1H, J=11 Hz, 2.3 Hz), 6.26 (s, 1H), 4.31 (s, 2H), 4.12 (m,2H), 3.63 (m, 4H), 2.33 (s, 3H), 1.51 (s, 9H), 1.45 (s, 9H).

8.N-[2-(Guanidinooxy)ethyl]-2-(2-methyl-5-{[benzylsulfonyl]-amino}phenyl)acetamideTrifluoroacetate Salt

The product of the preceding step (0.35 g, 0.56 mmol) was dissolved inDCM (10 mL) and treated with trifluoroacetic acid (3 mL) at ambienttemperature. After 16 hours the reaction was concentrated in vacuo andthe crude product purified on a 10 g Waters silica Sep-Pak® (5 to 20%methanol in DCM gradient elution) giving the title compound as a paleyellow solid (0.27 g, 89%). ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ 7.34 (m,3H), 7.29 (m, 2H), 7.14 (d, 1H, J=9.0 Hz), 7.02 (dd, 1H, J=6.5 Hz, 2.3Hz), 7.00 (s, 1H), 4.33 (s, 2H), 3.92 (br t, 2H, J=5 Hz), 3.55 (s, 2H),3.48 (br t, 2H, J=5 Hz), 2.28 (s, 3H). Mass spectrum (MALDI-TOF,gentisic acid matrix) calc'd. for C₁₉H₂₅N₅O₄S: 442.2 (M+Na), 420.2(M+H). Found: 442.5, 420.6.

Example 9N-[2-(Guanidinooxy)ethyl]-2-(2-hydroxy-6-methyl-3-{[(3-methylphenyl)sulfonyl]amino}phenyl)acetamideHydrochloride Salt

1. 4-Methyl-1-nitro-2-prop-2-enyloxybenzene

A solution of 5-methyl-2-nitrophenol (2.00 g, 13.1 mmol), allyl bromide(1.30 mL, 15.0 mmol), and cesium carbonate (5.5 g, 17 mmol) in DMF (100mL) was stirred at ambient temperature. After 20 hrs the reaction wasfiltered, frit washed with methanol, and the filtrate evaporated invacuo at 50° C. The residue was purified by flash column chromatography(4:1 then 2:1 hexane:ethyl acetate eluant) giving the title compound asa yellow oil (2.39 g, 95%) that crystallized after sitting 3 days atambient temperature. ¹H NMR (300 MHz, CDCl₃) δ 7.79 (d, 1H, J=8.2 Hz),6.86 (s, 1H), 6.82 (m, 1H), 6.05 (ddt, 1H, J=17.3 Hz, 10.6 Hz, 5.0 Hz),5.50 (ddd, 1H, J=17.3 Hz, 3.3 Hz, 1.7 Hz), 5.33 (ddd, 1H, J=10.6 Hz, 2.9Hz, 1.5 Hz), 4.67 (dt, 2H, J=4.9 Hz, 1.6 Hz), 2.40 (s, 3H).

2. 3-Methyl-6-nitro-2-prop-2-enylphenol

4-Methyl-1-nitro-2-prop-2-enyloxybenzene (7.11 g, 36.8 mmol), preparedas in the preceding step, was heated neat at 200° C. under nitrogen for3 hours, cooled to ambient temperature, and purified by flash columnchromatography (1:1 hexane:DCM eluant) giving the title compound as anorange oil (5.04 g, 71%). ¹H NMR (300 MHz, CDCl₃) δ 11.07 (s, 1H), 7.90(d, 1H, J=8.7 Hz), 6.80 (d, 1H, J=8.7 Hz), 5.93 (m, 1H), 5.03 (m, 1H),4.96 (m, 1H), 3.50 (dt, 2H, J=5.9 Hz, 1.7 Hz), 2.36 (s, 3H).

3. 1-Methyl-4-nitro-3-(phenylmethoxy)-2-prop-2-enylbenzene

A solution of the product of the preceding step (5.02 g, 26.0 mmol),benzyl bromide (3.40 mL, 28.6 mmol), and cesium carbonate (17.2 g, 52.8mmol) in DMF (100 mL) was stirred at ambient temperature for 20 hours.The solution was filtered, the filtrate concentrated in vacuo, and thecrude product adsorbed onto silica. This was poured onto a short bed ofsilica and eluted with DCM, and the eluate evaporated to give the titlecompound (7.20 g, 98%) as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.72(d, 1H, J=8.4 Hz), 7.46 (m, 2H), 7.37 (m, 3H), 7.06 (d, 1H, J=8.5 Hz),5.93 (m, 1H), 5.08 (m, 1H), 4.98 (s, 2H), 4.88 (m, 1H), 3.51 (dt, 2H,J=5.4 Hz, 1.9 Hz), 2.36 (s, 3H).

4. 4-Methyl-2-(phenylmethoxy)-3-prop-2-enylphenylamine

The product of the preceding step (0.55 g, 1.94 mmol) and tin(II)chloride dihydrate (2.89 g, 12.8 mmol) were stirred in reagent gradeethanol (40 mL) at ambient temperature. After 20 hours the reaction wasconcentrated in vacuo and the residue partitioned between saturatedsodium bicarbonate and DCM. The resulting emulsion was filtered, thesolids and aqueous layer washed with additional DCM, and the combinedorganic layers washed with brine, dried over sodium sulfate, andfiltered. The filtrate was then evaporated giving the title compound asan orange oil (0.51 g, 90%). ¹H NMR (300 MHz, CDCl₃) δ 7.47 (m, 2H),7.38 (m, 3H), 6.78 (d, 1H, J=8.1 Hz), 6.60 (d, 1H, J=8.0 Hz), 5.97 (m,1H), 5.02 (m, 1H), 4.95 (m, 1H), 4.85 (s, 2H), 3.64 (br s, 2H), 3.47(dt, 2H, J=5.7 Hz, 1.8 Hz), 2.20 (s, 3H).

5.[4-Methyl-2-(phenylmethoxy)-3-prop-2-enylphenyl][(3-methylphenyl)sulfonyl]amine

The product of the preceding step (0.49 g, 1.92 mmol) was dissolved inDCM (10 mL) and treated with m-toluenesulfonyl chloride (0.37 g, 1.96mmol) and N-methylmorpholine (0.25 mL, 2.27 mmol) at ambienttemperature. After 18 hours the reaction was concentrated in vacuo andthe residue purified by flash column chromatography (DCM eluant) givingthe title compound as a pale yellow oil (0.72 g, 92%). ¹H NMR (300 MHz,CDCl₃) δ 7.54 (m, 2H), 7.35 (m, 8H), 6.89 (d, 1H, J=8.3 Hz), 6.80 (s,1H), 5.87 (m, 1H), 4.99 (m, 1H), 4.79 (m, 1H), 4.43 (s, 2H), 3.36 (dt,2H, J=5.3 Hz, 1.9 Hz), 2.31 (s, 3H), 2.19 (s, 3H).

6.2-(6-Methyl-3-{[(3-methylphenyl)sulfonyl]amino}-2-(phenylmethoxy)phenyl)ethanol

The product of the preceding step (0.71 g, 1.74 mmol) was dissolved in1,4-dioxane (25 mL) and treated with a solution of sodium periodate(1.50 g, 7.01 mmol) in water (12 mL) and a 2.5 wt % solution of osmiumtetraoxide (0.25 mL, 0.02 mmol) in 2-methyl-2-propanol. After stirring 4hours at ambient temperature the reaction was diluted with DCM, washedwith 5% aqueous sodium bisulfite, water, and brine, dried over sodiumsulfate, and filtered. The filtrate was concentrated in vacuo giving apale yellow oil that was used without further purification.

7.2-(6-Methyl-3-{[(3-methylphenyl)sulfonyl]amino}-2-(phenylmethoxy)phenyl)aceticAcid

A solution of sodium dichromate (0.79 g, 2.64 mmol) and concentratedsulfuric acid (1.5 mL, 28 mmol) in water (25 ml) was added to a solutionof the product of the preceding step in acetone (25 mL), and thereaction stirred at ambient temperature for 3 days. After addingmethanol (3 mL) and stirring an additional 15 minutes, the organicsolvents were removed in vacuo and the remaining aqueous layer extractedwith DCM. The DCM layer was washed with brine, dried over sodiumsulfate, filtered, and the filtrate concentrated and purified by flashcolumn chromatography (10% methanol in DCM) giving the title compound asa pale yellow solid (0.51 g, 69% from Step 5). ¹H NMR (300 MHz, CDCl₃) δ7.56 (m, 2H), 7.39 (m, 4H), 7.31 (m, 4H), 6.93 (d, 1H, J=8.4 Hz), 6.78(s, 1H), 4.52 (s, 2H), 3.67 (s, 2H), 2.33 (s, 3H), 2.21 (s, 3H). Massspectrum (LCMS, ESI pos.) calc'd. for C₂₃H₂₃NO₅S: 448.1 (M+Na), 425.1(M+H). Found: 448.1, 425.9.

8.N-[2-({N,N′-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(6-methyl-3-{[(3-methylphenyl)sulfonyl]amino}-2-(phenylmethoxy)phenyl)acetamide

A solution of the product of the preceding step (0.32 g, 0.75 mmol), BOP(0.34 g, 0.77 mmol), triethylamine (0.25 mL, 1.80 mmol), and[N,N′-di(tert-butoxycarbonyl)]-2-aminoethoxyguanidine (Tianbao Lu, etal. WO 99/26926 (1999)) (0.27 g, 0.76 mmol), in DMF (15 mL) was stirredat ambient temperature overnight. The reaction was concentrated invacuo, the residue dissolved in DCM, washed with saturated sodiumbicarbonate, water, and brine, dried over sodium sulfate, and filtered.The filtrate was concentrated in vacuo and the crude product purified byflash column chromatography (7% methanol in DCM eluant) giving the titlecompound as a pale yellow solid (0.41 g, 76%). ¹H NMR (300 MHz, CDCl₃) δ9.11 (s, 1H), 7.93 (m, 1H), 7.55 (m, 3H), 7.36 (m, 8H), 6.90 (d, 1H,J=8.3 Hz), 6.84 (s, 1H), 4.60 (s, 2H), 4.06 (m, 2H), 3.71 (s, 2H), 3.56(dd, 2H, J=8.8 Hz, 5.2 Hz), 2.34 (s, 3H), 2.23 (s, 3H), 1.51 (s, 9H),1.43 (s, 9H).

9.N-[2-({N,N′-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(2-hydroxy-6-methyl-3-{[(3-methylphenyl)sulfonyl]amino}phenyl)acetamide

The product of the preceding step (0.41 g, 0.57 mmol) and 10% palladium(0) on carbon (60 mg) were dissolved in reagent grade ethanol (20 mL),degassed with nitrogen and vacuum, and stirred under a hydrogen balloonat ambient temperature for 4 hours. The reaction was filtered overCelite, the frit washed with methanol, the filtrate evaporated, and theresidue purified by preparative thin-layer chromatography (5% methanolin DCM eluant) giving the title compound as pale yellow solid (13.0 mg,4%). ¹H NMR (300 MHz, CDCl₃) δ 9.25 (s, [H), 8.62 (m, 1H), 7.61 (m, 3H),7.29 (m, 3H), 6.61 (d, 1H, J=8.5 Hz), 4.06 (m, 2H), 3.64 (s, 2H), 3.54(dd, 2H, J=8.7 Hz, 5.0 Hz), 2.34 (s, 3H), 2.30 (s, 3H), 1.51 (s, 9H),1.49 (s, 9H).

10.N-[2-(Guanidinooxy)ethyl]-2-(2-hydroxy-6-methyl-3-{[(3-methylphenyl)sulfonyl]amino}phenyl)acetamideHydrochloride Salt

The product of the preceding step (13.0 mg, 0.02 mmol) was dissolved inDCM (5 mL) and treated with trifluoroacetic acid (1 mL) at ambienttemperature. After 16 hours the reaction was concentrated in vacuo andthe crude product purified by preparative thin-layer chromatography (12%methanol in DCM eluant, saturated with ammonia). The resulting productwas treated with 4N HCl in ethanol, filtered, the filtrate evaporated,and the solid washed with diethyl ether and vacuum-dried giving thetitle compound (5.0 mg, 52%) as a tan solid. ¹H NMR (300 MHz, CD₃OD) δ7.51 (m, 2H), 7.38 (m, 2H), 6.56 (m, 2H), 3.91 (t, 2H, J=5.3 Hz), 3.61(s, 2H), 3.47 (t, 2H, J=5.3 Hz), 2.36 (s, 3H), 2.27 (s, 3H). Massspectrum (LCMS, ESI pos.) calc'd. for C₁₉H₂₅N₅O₅S: 436.1 (M+H). Found:436.2.

Example 10N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(2-hydroxy-6-methyl-3-{[(3-methylphenyl)sulfonyl]amino}phenyl)acetamideHydrochloride Salt

1.N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(6-methyl-3-{[(3-methylphenyl)sulfonyl]amino}-2-(phenylmethoxy)phenyl)acetamide

The product of Example 9, step 7 (0.18 g, 0.42 mmol), BOP (0.21 g, 0.47mmol), triethylamine (0.25 mL, 1.80 mmol), and2-amino-5-aminomethyl-6-methylpyridine dihydrochloride (Sanderson, P.E., et al. WO 97/01338 (1997)) (0.10 g, 0.48 mmol), were dissolved inDMF (10 mL) and stirred at ambient temperature for 2 hours. The reactionwas concentrated in vacuo and the crude product purified by flash columnchromatography (gradient elution: 10% to 15% methanol in DCM) giving animpure product that was dissolved in DCM, washed with saturated sodiumbicarbonate, water, and brine, dried over sodium sulfate, and filtered.The evaporated filtrate then gave the title compound as a pale yellowsolid (0.23 g, 99%). ¹H NMR (300 MHz, CDCl₃) δ 7.64 (m, 1H), 7.56 (m,1H), 7.35 (m, 10H), 7.11 (d, 1H, J=8.3 Hz), 6.94 (d, 1H, J=8.5 Hz), 6.22(d, 1H, J=8.1 Hz), 5.60 (br t, 1H, J=5.3 Hz), 4.49 (s, 2H), 4.37 (br s,2H), 4.19 (d, 2H, J=5.5 Hz), 3.56 (s, 2H), 2.34 (s, 3H), 2.23 (s, 3H),2.22 (s, 3H). Mass spectrum (LCMS, ESI pos.) calc'd. for C₃₀H₃₂N₄O₄S:545.2 (M+H). Found: 545.2.

2.N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(2-hydroxy-6-methyl-3-{[(3-methylphenyl)sulfonyl]amino}phenyl)acetamideHydrochloride Salt

The product of the preceding step (0.22 g, 0.41 mmol) and 10% palladium(0) on carbon (0.03 g) were dissolved in 2:1 ethanol:THF (30 mL),degassed with nitrogen and vacuum, and stirred under a hydrogen balloonat ambient temperature. After 7 hours the reaction was filtered overCelite, the frit washed with methanol, and the filtrate concentrated invacuo. The residue was treated with 4N HCl in ethanol (ca. 3 mL),evaporated under high vacuum, dissolved in DCM, filtered, and thefiltrate evaporated under high vacuum again giving the title compound(0.14 g, 71%) as a pale beige solid. ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ7.60 (m, 3H), 7.33 (m, 2H), 6.82 (d, 1H, J=8.2 Hz), 6.67 (d, 1H, J=9.0Hz), 6.58 (d, 1H, J=8.3 Hz), 4.18 (s, 2H), 3.58 (s, 2H), 2.45 (s, 3H),2.37 (s, 3H), 2.25 (s, 3H). Mass spectrum (LCMS, ESI pos.) calc'd. forC₂₃H₂₆N₄O₄S: 455.2 (M+H). Found: 455.2.

Example 113-({N-[2-(Guanidinooxy)ethyl]carbamoyl}methyl)-2-hydroxy-4-methylphenyl3-methylbenzenesulfonate Hydrochloride Salt

1. 2-Methoxy-4-methylphenyl 3-methylbenzenesulfonate

A solution of m-toluenesulfonyl chloride (0.53 g, 2.78 mmol) and2-methoxy-4-methylphenol (0.38 g, 2.75 mmol) in DCM (10 mL) was treatedwith triethylamine (0.5 mL, 3.6 mmol) and stirred at ambienttemperature. After 18 hours the reaction was concentrated in vacuo, theresidue dissolved in 1:1 hexane:DCM, filtered, and the filtrateevaporated under high vacuum giving the title compound as a white solid(0.79 g, 99%). ¹H NMR (300 MHz, CDCl₃) δ 7.72 (hr s, 1H), 7.66 (br d,1H, J=7.2 Hz), 7.45 (br d, 1H, J=7.2 Hz), 7.38 (t, 1H, J=7.6 Hz), 7.00(d, 1H, J=8.1 Hz), 6.67 (m, 1H), 3.54 (s, 3H), 2.42 (s, 3H), 2.31 (s,3).

2. 2-Hydroxy-4-methylphenyl 3-methylbenzenesulfonate

The product of the preceding step (0.79 g, 2.72 mmol) was dissolved inDCM (10 mL), cooled to −78° C., and treated with 1N boron tribromide inDCM (3.0 mL) under nitrogen. After 10 min the dry ice bath was removed,and the reaction stirred another hour while warming to ambienttemperature. After slowly quenching with water, the reaction was dilutedwith additional DCM, washed with brine, dried over sodium sulfate, andfiltered. The filtrate was concentrated in vacuo and the residuepurified by flash column chromatography (gradient elution: 50% to 33% to0% hexane in DCM) giving the title compound as a white crystalline solid(0.51 g, 68%). ¹H NMR (300 MHz, CDCl₃) δ 7.71 (br s, 1H), 7.67 (br d,1H, J=7.7 Hz), 7.51 (br d, 1H, J=8.1 Hz), 7.43 (t, 1H, J=7.7 Hz), 6.81(d, 1H J=1.71 Hz), 6.63 (d, 1H, J=8.3 Hz), 6.55 (m, 1H), 5.87 (s, 1H),2.43 (s, 3H), 2.26 (s, 3H).

3. 4-Methyl-2-prop-2-enyloxyphenyl 3-methylbenzenesulfonate

A solution of the product of the preceding step (0.51 g, 1.83 mmol),allyl bromide (0.20 mL, 2.30 mmol), and cesium carbonate (0.77 g, 2.40mmol) in DMF (25 mL) was stirred for 16 hours at ambient temperature.The reaction was concentrated in vacuo, the residue dissolved in DCM,filtered, and the filtrate washed with 1N aqueous KOH, water, and brine,dried over sodium sulfate, and filtered. The evaporated filtrate thengave the title compound as a pale yellow oil (0.54 g, 93%). ¹H NMR (400MHz, CDCl₃) δ 7.72 (s, 1H), 7.64 (d, 1H, J=7.7 Hz), 7.42 (d, 1H, J=7.7Hz), 7.35 (t, 1H, J=7.7 Hz), 7.04 (d, 1H, J=8.2 Hz), 6.68 (m, 2H), 5.80(ddt, 1H, J=17.3 Hz, 10.6 Hz, 5.1 Hz), 5.28 (ddd, 1H, J=17.3 Hz, 3.1 Hz,1.6 Hz), 5.20 (ddd, 1H, J=10.6 Hz, 2.8 Hz, 1.3 Hz), 4.29 (dt, 2H, J=5.1Hz, 1.5 Hz), 2.40 (s, 3H), 2.30 (s, 3H).

4. 2-Hydroxy-4-methyl-3-prop-2-enylphenyl 3-methylbenzenesulfonate

The product of the preceding step (0.54 g, 1.70 mmol) was heated neat at200° C. for 6 hrs, cooled to ambient temperature, and purified twice byflash column chromatography (first with 2:1 DCM:hexane, then with 4:1hexane:ethyl acetate eluant) giving the title compound as a colorlessoil (84 mg, 16%). ¹H NR (400 MHz, CDCl₃) δ 7.74 (s, 1H), 7.67 (d, 1H,J=7.8 Hz), 7.44 (d, 1H, J=7.6 Hz), 7.36 (t, 1H, J=7.7 Hz), 6.86 (d, 1H,J=8.3 Hz), 6.55 (d, 1H, J=8.3 Hz), 4.74 (m, 1H), 3.15 (dd, 1H, J=15.5Hz, 8.9 Hz), 2.61 (dd, 1H, J=15.5 Hz, 7.6 Hz), 2.41 (s, 3H), 2.16 (s,3H), 1.24 (d, 2H, J=6.3 Hz).

5. 4-Methyl-2-(phenylmethoxy)-3-prop-2-enylphenyl3-methylbenzenesulfonate

The product of the preceding step (68 mg, 0.21 mmol) and cesiumcarbonate (0.19 g, 0.58 mmol) were dissolved in DMF (5 mL) and treatedwith benzyl bromide (0.05 mL, 0.42 mmol) at ambient temperature. After 3days the reaction was concentrated in vacuo and the residue purified byflash column chromatography (DCM eluant) giving the title compound as apale yellow oil (50 mg, 58%). ¹HNMR (400 MHz, CDCl₃)δ 7.60 (br s, 1H),7.54 (br d, 1H, J=7.8 Hz), 7.34 (m, 4H), 7.27 (m, 3H), 7.10 (d, 1H,J=8.4 Hz), 6.91 (d, 1H, J=8.4 Hz), 5.74 (ddt, 1H, J=17.1 Hz, 10.2 Hz,5.7 Hz), 4.93 (dq, 1H, J=10.2 Hz, 1.7 Hz), 4.81 (s, 2H), 4.72 (dq, 1H,J=17.1 Hz, 1.8 Hz), 3.30 (dt, 2H, J=5.7 Hz, 1.7 Hz), 2.24 (s, 3H), 2.23(s, 3H).

6.4-Methyl-3-(2-oxoethyl)-2-(phenylmethoxy)phenyl-3-methylbenzenesulfonate

To a solution of the product of the preceding step (50 mg, 0.12 mmol)and sodium periodate (0.12 g, 0.56 mmol) in 5:1 acetonitrile water (12mL) was added ruthenium(III) chloride hydrate (8 mg, 0.04 mmol). Thereaction was stirred 6 hours at ambient temperature, diluted with DCM,and washed with 5% aqueous sodium bisulfite, water and brine. Theorganic solution was dried over sodium sulfate, filtered, and thefiltrate evaporated giving the title compound as a crude oil that wasused without further purification.

7.2-{6-Methyl-3-[(3-methylphenyl)sulfonyloxy]-2-(phenylmethoxy)phenyl]aceticAcid

The product of the preceding step was dissolved in acetone (5 mL) andtreated with a solution of sodium dichromate (65 mg, 0.22 mmol) andconcentrated sulfuric acid (1 mL) in water (4 mL) at ambienttemperature. After stirring 3 days the acetone was removed in vacuo, andthe remaining aqueous layer extracted with DCM. The organic phase wasthen washed with brine, dried over sodium sulfate, filtered, and theevaporated filtrate purified by flash column chromatography (8% methanolin DCM eluant) giving the title compound (45 mg, 88% from step 5) as awhite solid. ¹H NMR (400 MHz, CDCl₃) δ 7.61 (br s, 1H), 7.53 (br d, 1H,J=7.8 Hz), 7.33 (br d, 1H, J=7.6 Hz), 7.27 (m, 6H), 7.12 (d, 1H, J=8.4Hz), 6.92 (d, 1H, J=8.4 Hz), 4.87 (s, 2H), 3.60 (s, 2H), 2.24 (s, 3H),2.23 (s, 3H).

8.3-({N-[2-([N,N′-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]carbamoyl}methyl)-4-methyl-2-(phenylmethoxy)phenyl3-methylbenzenesulfonate

To a solution of the product of the preceding step (45 mg, 0.11 mmol),BOP (48 mg, 0.11 mmol), and[N,N′-di(tert-butoxycarbonyl)]-2-aminoethoxyguanidine (Tianbao Lu, etal., WO 99/26926 (1999)) (39 mg, 0.11 mmol), in DMF (5 mL) was addedtriethylamine (0.2 mL, 1.4 mmol). After stirring 18 hours at ambienttemperature, the reaction was concentrated in vacuo and the residuepurified by flash column chromatography (3:1 DCM:ethyl acetate eluant)giving the title compound as a colorless oil (61 mg, 79%). ¹H NMR (300MHz, CDCl₃) δ 9.11 (s, 1H), 7.65 (br s, 1H), 7.58 (br d, 1H, J=8.5 Hz),7.48 (m, 1H), 7.36 (br d, 1H, J=7.6 Hz), 7.29 (m, 7H), 7.03 (d, 1H,J=8.4 Hz), 6.90 (d, 1H, J=8.5 Hz), 4.92 (s, 2H), 4.02 (m, 2H), 3.66 (s,2H), 3.50 (dd, 2H, J=9.2 Hz, 5.2 Hz), 2.28 (s, 3H), 2.26 (s, 3H), 1.51(s, 9H), 1.45 (s, 9H).

9.3-({N-[2-({N,N′-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]carbamoyl}methyl)-2-hydroxy-4-methylphenyl3-methylbenzenesulfonate

The product of the preceding step (61 mg, 0.08 mmol) and 10% palladium(o) on carbon (20 mg) were dissolved in a 1:1:1 mixture of THF,methanol, and water (50 mL), degassed with nitrogen and vacuum, andstirred vigorously under a hydrogen balloon at ambient temperature.After 18 hours the reaction was filtered over Celite, the frit washedwith methanol, and the evaporated filtrate purified by flash columnchromatography (10% ethyl acetate in DCM eluant) giving the titlecompound as a colorless solid (40 mg, 74%). ¹H NMR (400 MHz, CDCl₃) δ9.25 (s, 1H), 8.50 (br t, 1H, J=4.9 Hz), 7.79 (s, 1H), 7.57 (d, 1H,J=7.7 Hz), 7.59 (br s, 1H), 7.43 (d, 1H, J=7.7 Hz), 7.38 (t, 1H, J=7.6Hz), 6.90 (d, 1H, J=8.3 Hz), 6.62 (d, 1H, J=8.4 Hz), 4.08 (m, 2H), 3.69(s, 2H), 3.56 (dd, 2H, J=8.7 Hz, 5.0 Hz), 2.41 (s, 3H), 2.35 (s, 3H),1.51 (s, 9H), 1.50 (s, 9H).

10.3-({N-[2-(Guanidinooxy)ethyl]carbamoyl}methyl)-2-hydroxy-4-methylphenyl3-methylbenzenesulfonate Hydrochloride Salt

The product of the preceding step (40 mg, 0.06 mmol) was dissolved inDCM (4 mL) and treated with neat trifluoroacetic acid (1.5 mL) atambient temperature. After 3 hours the reaction was concentrated invacuo and the residue purified by preparative thin-layer chromatography(20% methanol in DCM saturated with ammonia gas as eluant), treated with4N HCl in ethanol, and filtered. The evaporated filtrate was washed withdiethyl ether and dried under high vacuum giving the title compound as apale yellow solid (17 mg, 57%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.87 (s,1H), 9.76 (s, 1H), 7.67 (m, 5), 7.60 (d, 1H, J=7.6 Hz), 7.51 (t, 1H,J=7.7 Hz), 6.70 (d, 1H, J=8.3 Hz), 6.60 (d, 1H, J=8.5 Hz), 3.79 (t, 2H,J=5.5 Hz), 3.45 (m, 4H), 2.39 (s, 3H), 2.17 (s, 3H). Mass spectrum(LCMS, ESI pos.) calcd. for C₁₉H₂₄N₄O₆S: 437.1 (M+H). Found: 437.3.

Example 12 Tablet Preparation

Tablets containing 25.0, 50.0, and 100.0 mg, respectively, of thefollowing active compounds are prepared as illustrated below:

-   -   a.        N-[2-(Amidinoaminooxy)ethyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetamide        trifluoroacetate salt; and    -   b.        N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetamide        hydrochloride salt.

TABLET FOR DOSES CONTAINING FROM 25-100 MG OF THE ACTIVE COMPOUNDAmount-mg Active Compound 25.0 50.0 100.00 Microcrystalline cellulose37.25 100.0 200.0 Modified food corn starch 37.25 4.25 8.5 Magnesiumstearate 0.50 0.75 1.5

All of the active compound, cellulose, and a portion of the corn starchare mixed and granulated to 10% corn starch paste. The resultinggranulation is sieved, dried and blended with the remainder of the cornstarch and the magnesium stearate. The resulting granulation is thencompressed into tablets containing 25.0, 50.0, and 100.0 mg,respectively, of active ingredient per tablet.

Example 13 Intravenous Solution Preparation

An intravenous dosage form of the above-indicated active compounds ofExamples 1 and 2 is prepared as follows:

Active Compound 0.5-10.0 mg Sodium Citrate 5-50 mg Citric Acid 1-15 mgSodium Chloride 1-8 mg Water for Injection (USP) q.s. to 1 ml

Utilizing the above quantities, the active compound is dissolved at roomtemperature in a previously prepared solution of sodium chloride, citricacid, and sodium citrate in Water for Injection (USP, see page 1636 ofUnited States Pharmacopeia/National Formulary for 1995, published byUnited States Pharmacopeial Convention, Inc., Rockville, Md. (1994).

Example 14 In vitro Inhibition of Purified Enzymes

Reagents: All buffer salts were obtained from Sigma Chemical Company(St. Louis, Mo.), and were of the highest purity available. The enzymesubstrates, N-benzoyl-Phe-Val-Arg-p-nitroanilide (Sigma B7632),N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide hydrochloride (Sigma B2291),N-p-Tosyl-Gly-Pro-Lys-p-nitroanilide (Sigma T6140),N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma S7388) andN-CBZ-Val-Gly-Arg-p-nitroanilide (Sigma C7271) were obtained from Sigma.N-succinyl-Ala-Ala-Pro-Arg-p-nitroanilide (BACHEM L-1720) andN-succinyl-Ala-Ala-Pro-Val-p-nitroanilide (BACHEM L-1770) were obtainedfrom BACHEM (King of Prussia, Pa.).

Human α-thrombin, human factor Xa and human plasmin were obtained fromEnzyme Research Laboratories (South Bend, Ind.). Bovine α-chymotrypsin(Sigma C4129), bovine trypsin (Sigma T8642) and human kidney cellurokinase (Sigma U5004) were obtained from Sigma. Human leukocyteelastase was obtained from Elastin Products (Pacific, Mo.).

K_(i) Determinations: All assays are based on the ability of the testcompound to inhibit the enzyme catalyzed hydrolysis of a peptidep-nitroanilide substrate. In a typical K_(i) determination, substrate isprepared in DMSO, and diluted into an assay buffer consisting of 50 mMHEPES, 200 mM NaCl, pH 7.5. The final concentrations for each of thesubstrates is listed below. In general, substrate concentrations arelower than the experimentally determined value for K_(m). Test compoundsare prepared as a 1.0 mg/ml solution in DMSO. Dilutions are prepared inDMSO yielding 8 final concentrations encompassing a 200 foldconcentration range. Enzyme solutions are prepared at the concentrationslisted below in assay buffer.

In a typical K_(i) determination, into each well of a 96 well plate ispipetted 280 mL of substrate solution, 10 mL of test compound solution,and the plate allowed to thermally equilibrate at 37° C. in a MolecularDevices plate reader for >15 minutes. Reactions were initiated by theaddition of a 10 mL aliquot of enzyme and the absorbance increase at 405nm is recorded for 15 minutes. Data corresponding to less than 10% ofthe total substrate hydrolysis were used in the calculations. The ratioof the velocity (rate of change in absorbance as a function of time) fora sample containing no test compound is divided by the velocity of asample containing test compound, and is plotted as a function of testcompound concentration. The data are fit to a linear regression, and thevalue of the slope of the line calculated. The inverse of the slope isthe experimentally determined K_(i) value.

Thrombin: Thrombin activity was assessed as the ability to hydrolyze thesubstrate N-succinyl-Ala-Ala-Pro-Arg-p-nitroanilide. Substrate solutionswere prepared at a concentration of 32 mM (32 mM<<Km=180 mM) in assaybuffer. Final DMSO concentration was 4.3%. Purified human α-thrombin wasdiluted into assay buffer to a concentration of 15 nM. Final reagentconcentrations were: [thrombin]=0.5 nM, [substrateN-succinyl-Ala-Ala-Pro-Arg-p-nitroanilide]=32 nM.

Factor X [FXa]: FXa activity was assessed as the ability to hydrolyzethe substrate N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide hydrochloride.Substrate solutions were prepared at a concentration of 51 mM(51<<K_(m)=1.3 mM) in assay buffer. Final DMSO concentration was 4.3%.Purified activated human Factor X was diluted into assay buffer to aconcentration of 300 nM. Final reagent concentrations were: [FXa] 10 nM,[N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide hydrochloride]=51 mM.

Plasmin: Plasmin activity was assessed as the ability to hydrolyze theN-p-Tosyl-Gly-Pro-Lys-p-nitroanilide. Substrate solutions were preparedat a concentration of 37 mM (37 mM<<K_(m)=243 mM) in assay buffer. FinalDMSO concentration was 4.3%. Purified human plasmin was diluted intoassay buffer to a concentration of 240 nM. Final reagent concentrationswere: [Plasmin]=8 nM, [N-p-Tosyl-Gly-Pro-Lys-p-nitroanilide]=37 mM.

Chymotrypsin: Chymotrypsin activity was assessed as the ability tohydrolyze N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Substrate solutionswere prepared at a concentration of 14 mM (14 mM<<K_(m)=62 mM) in assaybuffer. Final DMSO concentration was 4.3%. Purified bovine chymotrypsinwas diluted into assay buffer to a concentration of 81 nM. Final reagentconcentrations were: [Chymotrypsin]=2.7 nM,[N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide]=14 mM.

Trypsin: Trypsin activity was assessed as the ability to hydrolyzeN-benzoyl-Phe-Val-Arg-p-nitroanilide. Substrate solutions were preparedat a concentration of 13 mM (13 mM<<K_(m)=291 mM) in assay buffer. FinalDMSO concentration was 4.3%. Purified bovine trypsin was diluted intoassay buffer to a concentration of 120 nM. Final reagent concentrationswere: [Trypsin]=4 nM, (N-benzoyl-Phe-Val-Arg-p-nitroanilide]=13 mM.

Elastase: Elastase activity was assessed as the ability to hydrolyzeN-succinyl-Ala-Ala-Pro-Val-p-nitroanilide. Substrate solutions wereprepared at a concentration of 19 mM (19 mM<<K_(m)=89 mM) in assaybuffer. Final DMSO concentration was 4.3%. Purified human leukocyteelastase was diluted into assay buffer to a concentration of 750 nM.Final reagent concentrations were: [Elastase]=25 nM,[N-succinyl-Ala-Ala-Pro-Val-p-nitroanilide]=19 mM.

Urokinase: Urokinase activity was assessed as the ability to hydrolyzeN-CBZ-Val-Gly-Arg-p-nitroanilide. Substrate solutions were prepared at aconcentration of 100 mM (100 mM<K_(m)=1.2 mM) in assay buffer. FinalDMSO concentration was 4.3%. Purified human kidney urokinase was dilutedinto assay buffer to a concentration of 1.2 mM. Final reagentconcentrations were: [Urokinase]=40 nM, and[N-CBZ-Val-Gly-Arg-p-nitroanilide]=100 mM.

The results indicate that the compounds of Examples 1 through 11 have Kivalues for human thrombin of between 0.0028 and 20 μM. The compound ofExample 5 has a Ki of 0.0028 μM.

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations, and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents and publications cited herein are fullyincorporated by reference herein in their entirety.

1. A compound of Formula I:

or a solvate, hydrate or pharmaceutically acceptable salt thereof;wherein: W is hydrogen, R¹, R¹OC(O), R¹C(O), R¹(CH₂)_(s)NHC(O), R¹S(O)₂,or (R¹)₂CH(CH₂)₅NHC(O), wherein s is 0-4; R¹ is R², R²(CH₂)_(t)C(R¹²)₂,where t is 0-3, and each R¹² can be the same or different,(R²)(OR¹²)CH(CH₂)_(p), where p is 1-4, (R²)₂(OR¹²)C(CH₂)_(p), where p is1-4, R²C(R¹²)₂(CH₂)_(t), wherein t is 0-3, and each R¹² can be the sameor different, wherein (R¹²)₂ can also form a ring with C represented byC₃₋₉ cycloalkyl, R²CF₂C(R¹²)₂(CH₂)_(q), wherein q is 0-2, and each R¹²can be the same or different, wherein (R¹²)₂ can also form a ring with Crepresented by C₃₋₉ cycloalkyl, R²CH₂C(R¹²)₂(CH₂)_(q), wherein q is 0-2,and each R¹² can be the same or different, wherein (R¹²)₂ can also forma ring with C represented by C₃₋₉ cycloalkyl, (R²)₂CH(CH₂)_(r), where ris 0-4 and each R² can be the same or different, and wherein (R²)₂ canalso form a ring with CH represented by C₃₋₉ cycloalkyl, C₇₋₁₂ bicylicalkyl, C₁₀₋₁₆ tricyclic alkyl, or a 5- to 7-membered mono- or bicyclicheterocyclic ring which can be saturated or unsaturated, and whichcontains from one to three heteroatoms selected from the groupconsisting of N, O and S, R²O(CH₂)_(p), wherein p is 2-4,(R²)₂CF(CH₂)_(r), wherein r is 0-4 and each R² can be the samedifferent, wherein (R²)₂ can also form a ring with C represented by C₃₋₉cycloalkyl, C₇₋₁₂ bicyclic alkyl, C₁₀₋₁₆ tricyclic alkyl, or a 5- to7-membered mono- or bicyclic heterocyclic ring which can be saturated orunsaturated, and which contains from one to three heteroatoms selectedfrom the group consisting of N, O and S,

where s is 0 or 1, or R²CF₂C(R¹²)₂; R² is phenyl, naphthyl, or biphenyl,each of which is unsubstituted or substituted with one or more of C₁₋₄alkyl, C₁₋₄ alkoxy, halogen, hydroxy, CF₃, OCF₃, COOH, CONH₂, or SO₂NH₂,a 5- to 7-membered mono- or a 9- to 10-membered bicyclic heterocyclicring or non-heterocyclic ring which can be saturated or unsaturated,wherein the heterocyclic ring contains from one to four heteroatomsselected from the group consisting of N, O and S, and wherein theheterocyclic or non-heterocyclic ring is unsubstituted or substitutedwith halogen or hydroxy, C₁₋₁₂ alky, unsubstituted or substituted withone or more of hydroxy, COOH, amino, optionally C₁₋₃ alkyl, substitutedaryl, C₃₋₉ cycloalkyl, CF₃, N(CH₃)₂, heteroaryl, or heterocycloalkyl,CF₃, C₃₋₉ cycloalkyl, unsubstituted or substituted with aryl, C₇₋₁₂bicyclic alkyl, or C₁₀₋₁₆ tricyclic alkyl; Y is —NH— or —O—; R³, R⁴, R⁵and R⁶ are independently hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl,optionally substituted aryl, optionally substituted aralkyl, optionallysubstituted heteroaryl, haloalkyl, hydroxy, alkoxy, aryloxy,heteroaryloxy, halogen, haloalkoxy, hydroxyalkyl, cyano, nitro,—CO₂R^(x), —CH₂OR^(x) or —OR^(x), where R^(x), in each instance, isindependently one of hydrogen, C₁₋₁₂ alkyl or C₃₋₉ cycloalkyl whereinsaid C-₁₂ alkyl or C₃₋₉ cycloalkyl groups may optionally have one ormore unsaturations; R¹¹ is hydrogen, alkyl, or alkenyl; R¹² is hydrogenor halogen, phenyl, naphthyl, or biphenyl, each of which isunsubstituted or substituted with one or more of C₁₋₄ alkyl, C₁₋₄alkoxy, halogen, hydroxy, CF₃, OCF₃, COOH, or CONH₂, a 5- to 7-memberedmono- or a 9- to 10-membered bicyclic heterocyclic ring which can besaturated or unsaturated, and which contains from one to fourheteroatoms selected from the group consisting of N, O and S, C₁₋₁₂alkyl, unsubstituted or substituted with one or more of hydroxy, COOH,amino, C₆₋₁₄ aryl, heteroaryl, or heterocycloalkyl, CF₃, C₃₋₉cycloalkyl, C₇₋₁₂ bicyclic alkyl, or C₁₀₋₁₆ tricyclic alkyl; B is:

wherein R⁹ and R¹⁰ are independently hydrogen, alkyl, aralkyl, aryl,hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl orcarboxyalkyl or R⁹ and R¹⁰ are taken together to form —(CH₂)_(n)—, wherev is 2 to 7, preferably 2 to 5; n is from zero to 8; and m is from zeroto 6; R¹⁴ and R¹⁵ are independently hydrogen, alkyl, cycloalkyl, halogenor alkoxy; and R¹⁶ and R¹⁷ are independently hydrogen, alkyl, hydroxy,alkoxy, aryloxy, alkoxycarbonyl, cyano or —CO₂R^(j), where R^(j) isC₁₋₁₂ alkyl, C₃₋₉ cycloalkyl, C₆₋₁₄ aryl, C₆₋₁₄ar(C₁₋₂)alkyl,halo(C₁₋₁₂)alkyl or

where R^(e), R^(f) and R^(g) are independently hydrogen or C₁₋₁₂ alkyl.2. A compound of claim 1, wherein R³, R⁴, R⁵ and R⁶ are independentlyhydrogen, C₁₋₁₂ alkyl, C₃₋₉ cycloalkyl, halogen, C₂₋₂₀ alkenyl, C₂₋₂₀alkynyl, optionally substituted C₆₋₁₄ aryl, optionally substitutedC₆₋₁₄ar(C₁₋₁₂)alkyl, optionally substituted heteroaryl,halo(C₁₋₁₂)alkyl, C₁₋₁₂ alkoxy, C₆₋₁₄ aryloxy, heteroaryloxy,halo(C₁₋₂₀)alkoxy or hydroxy(C₁₋₁₂)alkyl; R¹¹ is hydrogen, C₁₋₁₂ alkylor C₂₋₂₀ alkenyl; R⁹ and R¹⁰ are independently hydrogen, C₁₋₁₂ alkyl,C₆₋₁₄ar(C₁₋₂)alkyl, C₆₋₁₄ aryl, hydroxy(C₁₋₁₂)alkyl, amino(C₁₋₁₂)alkyl,mono(C₁₋₂)alkylamino(C₁₋₁₂)alkyl, di(C₁₋₁₂)alkylamino(C₁₋₁₂)alkyl, orcarboxy(C₁₋₂)alkyl; R¹⁴ and R¹⁵ are independently C₁₋₂ alkyl, C₃₋₉cycloalkyl or C₁₋₂₀ alkoxy; and R¹⁶ and R¹⁷ are independently C₁₋₁₂alkyl, C₁₋₂₀ alkoxy, C₆₋₁₄ aryloxy or C₁₋₂₀ alkoxycarbonyl.
 3. Acompound according to claim 1, wherein Y is —NH—.
 4. A compoundaccording to claim 1, wherein W is R¹ or R¹S(O)₂, where R¹ is R² and R²is either optionally substituted phenyl, naphthyl, biphenyl or C₁₋₇alkyl substituted with aryl, wherein the optional substituents on saidoptionally substituted phenyl, naphthyl, and biphenyl are selected fromthe group consisting of one or more of C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen,hydroxy, CF₃, OCF₃, COOH, CONH₂, or SO₂NH₂.
 5. A compound according toclaim 1, wherein W is R¹, where R¹ is R² or R²CF₂C(R¹²)₂(CH₂)_(q), andR² is either optionally substituted aryl or C₁₋₇ alkyl substituted witharyl; R¹² is hydrogen; and q is zero.
 6. A compound according to claim1, wherein R⁶ is C₁₋₆ alkyl or halogen.
 7. A compound according to claim6, wherein R⁶ is methyl, chloro or fluoro.
 8. A compound according toclaim 7, wherein R⁶ is chloro while R³ is fluoro or hydroxy.
 9. Acompound according to claim 1, wherein R¹¹ is hydrogen.
 10. A compoundaccording to claim 1, wherein each of R⁹ and R¹⁰ are hydrogen.
 11. Acompound of claim 1, which is one of:N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetamide;N-[(6-Amino-2,4-dimethyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetamideN-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)acetamideN-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(2-hydroxy-6-methyl-3-{[(3-methylphenyl)sulfonyl]amino}phenyl)acetamide;or or a solvate, hydrate or pharmaceutically acceptable salt thereof.12. A pharmaceutical composition, comprising a compound of claim 1 and apharmaceutically-acceptable carrier.
 13. A pharmaceutical composition,comprising a compound of claim 11 and a pharmaceutically-acceptablecarrier.
 14. A pharmaceutical composition according to claim 12, furthercomprising at least one of an anticoagulant, an antiplatelet agent or athrombolytic agent.
 15. A pharmaceutical composition according to claim12, wherein said compound is present in an amount between about 0.1 andabout 500 mg.
 16. A method of inhibiting or treating aberrantthrombosis, ischemia, stroke, restenosis or inflammation in a mammal inneed thereof, comprising administering to said mammal an effectiveamount of a compound of claim
 1. 17. A method of inhibiting or treatingthrombosis, ischemia, stroke, restenosis or inflammation in a mammal inneed thereof, comprising administering to said mammal an effectiveamount of a compound of claim
 11. 18. A method for the treatment ofstates characterized by abnormal venous or arterial thrombosis involvingeither thrombin production or action in a mammal in need thereof,comprising administering to said mammal a composition of claim
 12. 19. Amethod for the treatment of states characterized by abnormal venous orarterial thrombosis involving either thrombin production or action in amammal in need thereof, comprising administering to said mammal acomposition of claim
 13. 20. A method for the treatment of statescharacterized by abnormal venous or arterial thrombosis involving eitherthrombin production or action in a mammal in need thereof, comprisingadministering to said mammal a composition of claim
 14. 21. A method forthe treatment or prophylaxis of states characterized by abnormal venousor arterial thrombosis involving either thrombin production or action ina mammal in need thereof, comprising administering to said mammal acomposition of claim
 15. 22. A medical device for use in bloodcollection, blood storage or blood circulation, comprising a compound ofany claim 1 embedded in or physically attached to said medical device.23. A medical device according to claim 22, which is a catheter, stent,blood dialysis machine, blood collection syringe or tube, or a bloodline.
 24. A method of inhibiting the action of a proteolytic enzyme,wherein said proteolytic enzyme is a metalloprotease, acid protease,thiol protease or serine protease, comprising contacting said enzymewith a compound of claim
 1. 25. A method according to claim 24, whereinsaid enzyme is leukocyte neutrophil elastase, chymotrypsin, trypsin,urokinase, plasminogen activator, pancreatic elastase, cathepsin G,thrombin or factor Xa.
 26. A pharmaceutical composition according toclaim 12 adapted for oral administration.
 27. A pharmaceuticalcomposition according to claim 13 adapted for oral administration.
 28. Apharmaceutical composition according to claim 14 adapted for oraladministration.
 29. A pharmaceutical composition according to claim 15adapted for oral administration.
 30. A compound according to claim 1wherein v is 2 to 5.